Modified dicer polypeptide and methods of use thereof
View Patent ↗A modified Dicer polypeptide is provided, which modified Dicer polypeptide exhibits enhanced catalytic activity. Also provided is a method for producing small regulatory RNAs from a dsRNA, involving contacting a dsRNA with a subject modified Dicer. Small regulatory RNAs produced by a subject method find use in a variety of applications, including research and therapeutic applications.
1. A modified Dicer polypeptide, wherein said modified Dicer polypeptide lacks at least about 200 amino acids of the DExD/H-box domain set fourth in SEQ ID NO:2, generates miRNA or siRNA from a double-stranded RNA substrate, and exhibits a k cat that is at least 5-fold higher than the k cat of a Dicer polypeptide having the amino acid sequence set fourth in SEQ ID NO:1.
2. The modified Dicer polypeptide of claim 1 , wherein the modified Dicer polypeptide lacks the DExD/H-box domain.
3. The modified Dicer polypeptide of claim 2 , wherein the modified Dicer polypeptide lacks an amino acid sequence having at least about 75% amino acid sequence identity with a stretch of at least 100 contiguous amino acids of amino acids 1-604 of the amino acid sequence set fourth in SEQ ID NO:2.
4. The modified Dicer polypeptide of claim 1 , wherein the modified Dicer polypeptide comprises an amino acid sequence having at least about 75% amino acid sequence identity to the amino acid sequence set fourth in SEQ ID NO:3.
5. The modified Dicer polypeptide of claim 1 , wherein said modified Dicer polypeptide has a k cat of from about 0.5×10 −1 s −1 to about 1.0×10 −5 s −1 .
6. A composition comprising: a) the modified Dicer polypeptide of claim 1 ; and b) a buffer.
7. A nucleic acid comprising a nucleotide sequence encoding a subject modified Dicer polypeptide of claim 1 .
8. The nucleic acid of claim 7 , wherein said nucleic acid is a recombinant expression construct.
9. A genetically modified host cell comprising the nucleic acid of claim 7 .
10. The genetically modified host cell of claim 9 , wherein said host cell is a prokaryotic host cell.
11. The genetically modified host cell of claim 9 , wherein said host cell is a eukaryotic host cell.
12. A method of producing the modified Dicer polypeptide of claim 1 , the method comprising:
a) culturing the genetically modified host cell of claim 9 in culture medium in vitro under conditions such that the cell produces the modified Dicer polypeptide; and
b) recovering the modified Dicer polypeptide produced by the cell.
13. A method of producing an siRNA, the method comprising contacting the modified Dicer polypeptide of claim 1 with a double-stranded RNA (dsRNA) substrate, wherein the modified Dicer polypeptide cleaves the dsRNA substrate, thereby producing an siRNA.
14. The method of claim 13 , wherein the siRNA has a length of from about 21 to about 23 nucleotides.
15. The modified Dicer polypeptide of claim 1 , wherein the modified Dicer polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:3.
16. The modified Dicer polypeptide of claim 15 , wherein the modified Dicer polypeptide has a length of from about 1100 amino acids to about 1320 amino acids.
17. The modified Dicer polypeptide of claim 15 , wherein the modified Dicer polypeptide exhibits a k cat that is at least 10-fold higher than the k cat of a Dicer polypeptide having the amino acid sequence set forth in SEQ ID NO:1.
18. The modified Dicer polypeptide of claim 15 , wherein the modified Dicer polypeptide exhibits a k cat that is at least 40-fold higher than the k cat of a Dicer polypeptide having the amino acid sequence set forth in SEQ ID NO:1.