Nucleic acids for down-regulation of gene expression
Recombinant nucleic acid molecules are provided that form hair pin structures and can be used to down-regulate gene expression. For example, a nucleic acid molecule can comprise a flanking and lower stem loop sequence from a mir-16 gene; an antisense target sequence; a mir-30 loop sequence; a complement of the anti-sense target sequence; and a lower stem loop complementary to the mir-16 sequence. Methods for down regulating gene expression in a cell using such recombinant nucleic acid molecules are also provided.
1. A recombinant nucleic acid molecule comprising from 5′ to 3′ and in the order from (a)-(g):
(a) a flanking sequence;
(b) a first lower stem sequence which is 12, 13, 14, 15 or 16 nucleotides in length;
(c) an anti-sense target sequence 22 nucleotides in length;
(d) a loop sequence;
(e) a sense sequence wherein the sequence is complementary to the sequence of (c) except that the sequence comprises one or two mismatches relative to the sequence of (c), wherein the one or two mismatches comprise:
i) a mismatch located at the position 8 to 14 of the sense sequence; or
ii) a mismatch at the final 3′ position (position 22) of the sense sequence;
(f) a second lower stem sequence wherein the sequence is complementary to the sequence of (b); and
(g) a second flanking sequence.
2. The nucleic acid molecule of claim 1 , wherein the lower stem is 13, 14, 15 or 16 nucleotides in length.
3. The nucleic acid molecule of claim 1 , wherein the sense sequence (e) comprises one mismatch relative to sequence (c) located at nucleotide position 11 of the sense sequence (e).
4. The nucleic acid molecule of claim 1 , wherein the sense sequence (e) comprises two mismatches relative to sequence (c) located (i) at position 11 of the sense sequence (e) and (ii) at the last 3′ nucleotide (position 22) of the sense sequence (e).
5. The nucleic acid molecule of claim 1 , comprising a flanking sequence (g), wherein the flanking sequence is not complementary to the flanking sequence (a).
6. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule is RNA.
7. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule is DNA.
8. The nucleic acid molecule of claim 1 , wherein the flanking sequence (a) comprises a mir-16 sequence.
9. The nucleic acid molecule of claim 1 , wherein the first lower stem sequence (b) comprises a mir-16 sequence.
10. The nucleic acid molecule of claim 9 , wherein the first lower stem (b) comprises the mir-16 sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
11. The nucleic acid molecule of claim 1 , wherein the mir-16 flanking sequence (a) comprises the sequence of SEQ ID NO: 1.
12. The nucleic acid molecule of claim 1 , wherein the loop sequence comprises a mir-30 sequence of SEQ ID NO: 7.
13. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule comprises at least 2 repeats of the sequences (a)-(g).
14. The nucleic acid molecule of claim 1 , comprising from 5′ to 3′ and in the order from (a)-(g):
(a) a mir-16 flanking sequence;
(b) a first lower stem sequence comprising a mir-16 sequence;
(c) an anti-sense target sequence 22 nucleotides in length;
(d) a mir-30 loop sequence;
(e) a sense sequence wherein the sequence is complementary to the sequence of (c) except that the sequence comprises one or two mismatches relative to the sequence of (c), wherein the one or two mismatches comprise:
i) a mismatch located at the position 8 to 14 of the sense sequence; or
ii) a mismatch at the final 3′ position (position 22) of the sense sequence;
(f) a second lower stem sequence wherein the sequence is complementary to the sequence of (b), wherein the lower stem is at least 11 nucleotides in length; and
(g) a second flanking sequence.
15. The nucleic acid molecule of claim 14 , comprising from 5′ to 3′ and in the order from (a)-(g):
(a) a mir-16 flanking sequence comprising the sequence of SEQ ID NO: 1;
(b) a first lower stem sequence comprising the mir-16 sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;
(c) an anti-sense target sequence 22 nucleotides in length;
(d) a mir-30 loop sequence comprising the sequence of SEQ ID NO: 7;
(e) a sense sequence wherein the sequence is complementary to the sequence of (c) except that the sequence comprises one or two mismatches relative to the sequence of (c), wherein the one or two mismatches comprise:
i) a mismatch located at the position 8 to 14 of the sense sequence; or
ii) a mismatch at the final 3′ position (position 22) of the sense sequence;
(f) a second lower stem sequence wherein the sequence is complementary to the sequence of (b); and
(g) a second flanking sequence.
16. The nucleic acid molecule of claim 1 , wherein the anti-sense target sequence is complementary to a CCR5 mRNA sequence.
17. An expression vector comprising a nucleic acid of 1 molecule of claim 1 operably linked to a promoter sequence.
18. The expression vector of claim 17 , wherein the promoter is an inducible, tissue-specific-or cell lineage-specific promoter.
19. The expression vector of claim 17 , further comprising at least one drug resistance marker.
20. A host cell comprising the nucleic acid molecule of claim 1 .
21. A method for reducing expression of a gene in a cell comprising expressing a nucleic acid molecule of any one of claim 1 in the cell wherein the anti-sense target sequence (c) is complementary to the sense strand of the gene.