Micro-RNA scaffolds, non-naturally occurring micro-RNAs, and methods for optimizing non-naturally occurring micro-RNAs
The present disclosure provides non-naturally occurring miR-196a-2 miRNAs and non-naturally occurring miR-204 miRNAs. The non-naturally occurring miRNAs of the disclosure have mature strand sequences distinct from their endogenous counterparts. The disclosure also provides methods of selecting mature strand sequences that function optimally in non-naturally occurring miR-196a-2 miRNAs. 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 wherein the stem-loop structure incorporates a miR-196a-2 scaffold and a mature strand and star strand duplex, wherein the sequence of said mature strand of the duplex is distinct from the sequence of the endogenous mature strand of any miRNA and is either: (i) 100% complementary to a portion of a target RNA; or (ii) 100% complementary to a portion of the target RNA except that the target RNA is capable of forming a wobble base pair with position 1 of the mature strand, and wherein said star strand is complementary to said mature strand except that 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, or mismatches or wobble pairs at one or more of positions 5, 18, 19, 20, 21, 22, or 23 and no mismatches or wobble pairs at any of positions 2, 3, 4, 6-11 and 13-17 of the mature strand of the duplex, wherein positions of the mature strand are numbered relative to the 5′ end of the mature strand and wherein said mature strand of the duplex is between about 19 and 25 nucleotides in length.
2. The non-naturally occurring miR-196a-2 miRNA of claim 1 , wherein the nucleic acid comprises the sequence (SEQ ID NO: 10):
wherein M is said mature strand of the duplex and wherein S is said star strand.
3. The non-naturally occurring miR-196a-2 miRNA of claim 1 , wherein said mature strand of the duplex is about 19 nucleotides in length.
4. 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-196-a-2 scaffold and a mature strand-star strand duplex, wherein the sequence of said mature strand of the duplex is distinct from the sequence of the endogenous mature strand of any miRNA and is either: (i) 100% complementary to a portion of a target RNA; or (ii) 100% complementary to a portion of the target RNA except that the target RNA is capable of forming a wobble base pair with position 1 of the mature strand, and wherein said star strand is complementary to said mature strand of the duplex except that 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, or mismatches or wobble pairs at one or more of positions 5, 18, 19, 20, 21, 22 or 23 and no mismatches or wobble pairs at any of positions 2, 3, 4, 6-11 and 13-17, wherein positions of the mature strand are numbered relative to the 5′ end of the mature strand, and wherein said mature strand of the duplex is between about 19 and 25 nucleotides in length.
5. The recombinant expression vector of claim 4 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.
6. The recombinant expression vector of claim 5 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.
7. The recombinant expression vector of claim 4 , wherein said vector is a lentiviral vector.
8. 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-196-a-2 scaffold and a mature strand-star strand duplex, wherein the sequence of said mature strand of the duplex is distinct from the sequence of the endogenous mature strand of any miRNA and is either: (i) 100% complementary to a portion of a target RNA; or (ii) 100% complementary to a portion of the target RNA except that the target RNA is capable of forming a wobble base pair with position 1 of the mature strand, and wherein said star strand is complementary to said mature strand of the duplex except that 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, or mismatches or wobble pairs at one or more of positions 5, 18, 19, 20, 21, 22 or 23 and no mismatches or wobble pairs at any of positions 2, 3, 4, 6-11 and 13-17 of the mature strand of the duplex, wherein positions of the mature strand are numbered relative to the 5′ end of the mature strand and wherein said mature strand of the duplex is between about 19 and 25 nucleotides in length; and at least one pharmaceutically acceptable carrier.
9. The non-naturally occurring miRNA of claim 1 , wherein said non-naturally occurring miRNA comprises a mismatch or wobble pair at one or more of positions 5, 18, 19, 20 or 21 of the mature strand and each position is numbered relative to the 5′ end of the mature strand.
10. The non-naturally occurring miRNA of claim 9 , wherein said non-naturally occurring miRNA comprises a mismatch or wobble pair at position 5 of the mature strand, wherein the position is numbered relative to the 5′ end of the mature strand.