Tripartite oligonucleotide complexes and methods for gene silencing by RNA interference
Provided herein are tripartite oligonucleotide complexes which can be administered to a cell, tissue or organism to silence a target gene. The tripartite oligonucleotide complexes of the disclosure may include a conjugate moiety that facilitates delivery to a cell, tissue or organism without the aid of a transfection reagent.
1. A tripartite oligonucleotide complex comprising:
i) a first oligonucleotide between 29 nucleotides and 37 nucleotides in length having a target-dependent region and a target-independent region that is 5′ to the target-dependent region,
ii) a second oligonucleotide between 19 nucleotides and 23 nucleotides in length comprising an antisense region that has greater than 70% complementarity to a target gene of an organism, and
iii) a third oligonucleotide between 10 nucleotides and 14 nucleotides in length,
wherein the target-dependent region of the first oligonucleotide is greater than 80% complementary to the second oligonucleotide and the target-independent region of the first oligonucleotide is greater than 80% complementary to the third oligonucleotide, such that the first, second and third oligonucleotides are capable of forming a tripartite complex having at least two regions of duplex, and the third oligonucleotide is a non-targeting sequence.
2. The tripartite oligonucleotide complex of claim 1 wherein the first oligonucleotide has a 3′ overhang of 1-6 nucleotides.
3. The tripartite oligonucleotide complex of claim 2 wherein the first oligonucleotide has a 3′ overhang of 2 nucleotides.
4. The tripartite oligonucleotide complex of claim 3 wherein the first oligonucleotide is 37 nucleotides in length including said 3′ overhang of 2 nucleotides, the second oligonucleotide is 21 nucleotides in length including a UU dinucleotide sequence at the 3′ end, and the third oligonucleotide is 14 nucleotides in length.
5. The tripartite oligonucleotide complex of claim 1 wherein about 40% to about 90% of all nucleotides in each of the first, second and third oligonucleotides are chemically modified and wherein the 5′ terminus of the second oligonucleotide is phosphorylated.
6. The tripartite oligonucleotide complex of claim 5 wherein all the Us and Cs in the first oligonucleotide are 2′ O-methyl modified, all Us and Cs of the second oligonucleotide are 2′ F modified, and all the nucleotides of the third oligonucleotide are 2′ O-methyl modified.
7. The tripartite oligonucleotide complex of claim 4 wherein nucleotide positions in the target-dependent region of the first oligonucleotide that are opposite nucleotide positions 18 and 19 of the second oligonucleotide counting from the 5′ end are 2′ O-methyl modified.
8. The tripartite oligonucleotide complex of claim 4 wherein a mismatch exists between position 10, 11, 12, 13, or 14 on the second oligonucleotide counting from the 5′ end and the opposite nucleotide in the target-dependent region of the first oligonucleotide.
9. The tripartite oligonucleotide complex of claim 8 wherein a mismatch exists between position 14 on the second oligonucleotide counting from the 5′ end and the opposite nucleotide in the target-dependent region of the first oligonucleotide.
10. The tripartite oligonucleotide complex of claim 2 wherein the sequence of the boundary between the target-independent region of the first oligonucleotide and the target-dependent of region of the first oligonucleotide is 5′ G/AA 3′.
11. The tripartite oligonucleotide complex of claim 2 further comprising at least one conjugate moiety attached to the third oligonucleotide.
12. The tripartite oligonucleotide complex of claim 11 wherein said conjugate moiety is attached to the 3′ terminus of the third oligonucleotide.
13. The tripartite oligonucleotide complex of claim 12 wherein said conjugate moiety is attached to the 3′ terminus of the third oligonucleotide via a linker.
14. The tripartite oligonucleotide complex of claim 13 wherein said conjugate moiety is selected from the group consisting of a steroid molecule, a vitamin, and a peptide.
15. The tripartite oligonucleotide complex of claim 14 wherein said conjugate moiety is a steroid molecule selected from the group consisting of cholesterol, cholestanol, stigmasterol, cholanic acid, and ergosterol.
16. The tripartite oligonucleotide complex of claim 15 wherein said conjugate moiety is cholesterol, wherein said linker is a C5 linker molecule, and wherein the third oligonucleotide has the structure:
17. The tripartite oligonucleotide complex of claim 2 further comprising at least one detectable label attached to the third oligonucleotide.
18. The tripartite oligonucleotide complex of claim 17 further comprising at least one conjugate moiety.
19. The tripartite oligonucleotide of claim 17 wherein said detectable label is a dye molecule.
20. A pharmaceutical composition comprising a tripartite oligonucleotide complex, said tripartite oligonucleotide complex comprising:
i) a first oligonucleotide between 29 nucleotides and 37 nucleotides in length having a target-dependent region and a target-independent region that is 5′ to the target-dependent region,
ii) a second oligonucleotide between 19 nucleotides and 23 nucleotides in length comprising an antisense region that has greater than 70% complementarity to a target gene of an organism, and
iii) a third oligonucleotide between 10 nucleotides and 14 nucleotides in length,
wherein the target-dependent region of the first oligonucleotide is greater than 80% complementary to the second oligonucleotide nucleotide and the sequence of the target-independent region of the first oligonucleotide is greater than 80% complementary to the third oligonucleotide, such that the first, second and third oligonucleotides are capable of forming a tripartite complex having at least two regions of duplex, and the third oligonucleotide is a non-targeting sequence.
21. The pharmaceutical composition of claim 20 wherein the first oligonucleotide is 37 nucleotides in length and includes a 3′ overhang of 2 nucleotides, the second oligonucleotide is 21 nucleotides in length including a UU dinucleotide sequence at the 3′ end, and the third oligonucleotide is 14 nucleotides in length.
22. The pharmaceutical composition of claim 20 wherein about 40% to about 90% of all nucleotides in each of the first, second and third oligonucleotides are chemically modified and wherein the 5′ terminus of the second oligonucleotide is phosphorylated.
23. The pharmaceutical composition of claim 21 wherein nucleotide positions in the target-dependent region of the first oligonucleotide that are opposite nucleotide positions 18 and 19 of the second oligonucleotide counting from the 5′ end are 2′ O-methyl modified.
24. The pharmaceutical composition of claim 23 wherein a mismatch exists between position 10, 11, 12, 13, or 14 on the second oligonucleotide counting from the 5′ end and the opposite nucleotide in the target-dependent region of the first oligonucleotide.
25. The pharmaceutical composition of claim 20 wherein said tripartite oligonucleotide complex further comprises at least one conjugate moiety attached to the third oligonucleotide.