Micro-RNA scaffolds and non-naturally occurring micro-RNAs
The present disclosure provides a non-naturally occurring miRNA having a stem-loop structure comprising a scaffold derived from a first endogenous miRNA (e.g., miR-196a-2 or miR-204), a mature strand derived from a second endogenous miRNA, and a star strand sequence that is at least partially complementary to the mature strand sequence. The present disclosure also provides a non-naturally occurring miRNA having a stem-loop structure comprising a scaffold derived from an endogenous miRNA (e.g., miR-196a-2 or miR-204), a mature strand designed t be at least partially complementary to a target RNA, and a star strand sequence that is at least partially complementary to the mature strand sequence. The methods and compositions of the disclosure may be used to mediate gene silencing via the RNAi pathway.
1. A non-naturally occurring miR-196a-2 miRNA comprising a nucleic acid having a stem-loop structure comprising a miR-196a-2 scaffolding and a mature strand-star strand duplex, wherein the sequence of said mature strand of said duplex is derived from a mature endogenous miRNA but is distinct from the sequence of the endogenous mature strand of miR-196a-2, and wherein position 1 of the mature strand is U and the opposite nucleotide in the star strand is G and the duplex contains a mismatch at position 12 of the mature strand and either no additional mismatches or wobble pairs at any position other than positions 1 and 12 of the mature strand, or mismatches or wobble pairs at one or more of positions 5, 18, 19, 20, 21, 22 or 23 of the mature strand of the duplex and no mismatches or wobble pairs at any of positions 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16 and 17 within the duplex, wherein positions of the mature strand are numbered relative to the 5′ end of the mature strand, the non-naturally occurring miR-196a-2 is a non-naturally occurring miRNA, and the mature strand is 19-25 nucleotides in length.
2. The non-naturally occurring miR-196a-2 miRNA of claim 1 wherein the sequence of the loop is the same as the sequence of the loop from endogenous miR-196a-2.
3. The non-naturally occurring miR-196a-2 miRNA of claim 1 wherein said stem-loop structure comprises the sequence (SEQ ID NO: 10):
wherein M is said mature strand and wherein S is said star strand.
4. The non-naturally occurring miR-196a-2 miRNA of claim 1 , wherein said mature strand of the duplex is 19-23 nucleotides in length.
5. A cell comprising the non-naturally occurring miR-196a-2 miRNA of claim 1 .
6. The non-naturally occurring miRNA of claim 1 further comprising a mismatch or wobble pair at one or more of positions 5, 18, 19, 20, and 21 of the mature strand.
7. The non-naturally occurring miRNA of claim 6 , wherein said non-naturally occurring miRNA comprises a mismatch or wobble pair at position 5 of the mature strand.
8. A recombinant expression vector comprising a nucleotide sequence that encodes a non-naturally occurring miR-196a-2 miRNA, said non-naturally occurring miR-196a-2 miRNA comprising a nucleic acid having a stem-loop structure wherein the stem-loop structure incorporates a miR-196a-2 scaffold and a mature strand-star strand duplex, wherein
the sequence of said mature strand of the duplex is derived from a mature endogenous miRNA but is distinct from the sequence of the endogenous mature strand of miR-196a-2, and wherein position 1 of the mature strand is U and the opposite nucleotide in the star strand is G and the duplex contains a mismatch at position 12 of the mature strand and either no additional mismatches or wobble pairs, at any position other than positions 1 and 12 of the mature strand, or mismatches or wobble pairs at one or more of positions 5, 18, 19, 20, 21, 22, or 23 of the mature strand of the duplex, and no mismatches or wobble pairs at positions 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16 and 17 within the duplex, wherein positions of the mature strand are numbered relative to the 5′ end of the mature strand and the mature strand is 19-25 nucleotides in length.
9. The recombinant expression vector of claim 8 wherein said vector comprises a promoter operably linked to a reporter gene comprising an artificial intron, and wherein said non-naturally occurring miRNA is located within said artificial intron.
10. The recombinant expression vector of claim 8 wherein said vector comprises a promoter operably linked to a reporter gene having a 3′ untranslated region (UTR), and wherein said non-naturally occurring miRNA is located within said 3′ UTR.
11. The recombinant expression vector of claim 8 , wherein said vector is a lentiviral vector.
12. A pharmaceutical composition comprising:
a non-naturally occurring miR-196a-2 miRNA, said non-naturally occurring miR-196a-2 miRNA comprising a nucleic acid having a stem-loop structure wherein the stem-loop structure incorporates a miR-196a-2 scaffold and a mature strand-star strand duplex, wherein the sequence of said mature strand of the duplex is derived from a mature endogenous miRNA but is distinct from the sequence of the endogenous mature strand of miR-196a-2, and wherein position 1 of the mature strand is U and the opposite nucleotide in the star strand is G and the duplex contains a mismatch at position 12 of the mature strand and either no additional mismatches or wobble pairs, at any position other than positions 1 and 12 of the mature strand, or mismatches or wobble pairs at one or more of positions 5, 18, 19, 20, 21, 22, or 23 of the mature strand of the duplex, and no mismatches or wobble pairs at positions 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16 and 17 within the duplex, wherein positions of the mature strand are numbered relative to the 5′ end of the mature strand and at least one pharmaceutically acceptable carrier and the mature strand is 19-25 nucleotides in length.