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 to 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 method of delivery of a mature endogenous miRNA to a cell, the method comprising:
introducing in said cell a non-naturally occurring miR-196a-2 miRNA, wherein said non-naturally occurring miR-196a-2 miRNA comprises a nucleic acid having a stem-loop structure, wherein the stem of the stem-loop structure incorporates a miRNA-196-a-2 scaffold, and a mature strand-star strand duplex, wherein said mature strand has a sequence derived from a mature endogenous miRNA, wherein said mature endogenous miRNA has a sequence that is distinct from the sequence of the endogenous mature strand of miR-196a-2, 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 other than 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 about 19-25 nucleotides in length;
wherein said non-naturally occurring miR-196a-2 miRNA is processed in said cell to yield a mature miRNA having the sequence of said mature strand.
2. The method of claim 1 , wherein said non-naturally occurring miR-196a-2 comprises (SEQ ID NO: 10):
wherein M is said mature strand and wherein S is said star strand.
3. The method of claim 1 , wherein said introducing comprises contacting the cell with a vector capable of expressing the non-naturally occurring miR-196a-2 miRNA.
4. The method of claim 3 , wherein the vector is a lentiviral vector.
5. The method of claim 1 , wherein said introducing comprises providing a recombinant expression vector, wherein the expression vector comprises a nucleotide sequence that encodes the non-naturally occurring miR-196a-2 miRNA.
6. The method of claim 1 , wherein the mature strand is 19-23 nucleotides in length.
7. The method of claim 1 , wherein the non-naturally occurring miR-196a-2 miRNA comprises a mismatch or wobble pair at one or more of positions 5, 18, 19, 20, or 21 of the mature strand.
8. The method of claim 1 , wherein the nucleotide at position 12 of the mature strand is the same as the nucleotide at the opposite position of the star strand.
9. The method of claim 5 , wherein the nucleotide at position 12 of the mature strand is the same as the nucleotide at the opposite position of the star strand.
10. The method of claim 6 , wherein the nucleotide at position 12 of the mature strand is the same as the nucleotide at the opposite position of the star strand.
11. The method of claim 7 , wherein the nucleotide at position 12 of the mature strand is the same as the nucleotide at the opposite position of the star strand.
12. The method of claim 1 , wherein said introducing is by transfection.