Short/small hairpin RNA molecules

12065,647

16/971893

February 22, 2019

This disclosure concerns compositions and methods for novel RNA inhibition molecules (e.g., small/short hairpin RNA (shRNA) molecules), polynucleotides encoding such molecules, use of such novel RNA inhibition molecules to inhibit a target gene by suppressing the expression of the mRNA of the target gene, and for application such as the of control insect pests, and transgenic plants that produce, and are protected, by these novel RNA inhibition molecules are described.

DOW AGROSCIENCES LLC (Indianapolis, IN, US); QUEENSLAND UNIVERSITY OF TECHNOLOGY (Brisbane, AU)

DOW AGROSCIENCES LLC (Indianapolis, IN, US), QUEENSLAND UNIVERSITY OF TECHNOLOGY (Brisbane, AU)

1. A small hairpin RNA (shRNA) polynucleotide less than 70 nucleotides in length, the polynucleotide comprising the following structures: a) a hairpin structure consisting of less than or equal to 25 nucleotides, obtained from an insect microRNA scaffold, that comprises double-stranded nucleotides and single-stranded nucleotides; b) a DICER-1 recognition and cleavage site structure; and c) a double-stranded stem structure consisting of 16-25 nucleotide pairs, wherein the two strands of the double-stranded stem structure are 100% complementary to one another; wherein the two strands of the double-stranded stem structure (c) are connected at one end of the double-stranded stem structure via the hairpin structure (a); wherein the stem structure comprises a polynucleotide that shares 70% to 100% sequence identity with a target polynucleotide of a plant pest; wherein the shRNA is unable to be cleaved by insect DROSHA.

2. The shRNA polynucleotide of claim 1 , wherein the shRNA comprises 2 alpha-helical turns.

3. The shRNA polynucleotide of claim 1 , wherein the shRNA is not recognized by insect DROSHA.

4. The shRNA polynucleotide of claim 1 , wherein the hairpin structure comprises mismatches and/or bulges.

5. The shRNA polynucleotide of claim 1 , wherein the insect microRNA scaffold is SEQ ID NO:7.

6. The shRNA polynucleotide of claim 1 , wherein the target polynucleotide of a plant pest is selected from the group consisting of: COPI gamma, COPI alpha, COPI beta, and COPI delta.

7. The shRNA polynucleotide of claim 1 , wherein the shRNA has a free energy (ΔG° 37) of less than −30 kcal/mol.

8. The shRNA polynucleotide of claim 1 , wherein the shRNA further comprises a polynucleotide overhang of at least 2 nucleotides and not more than 4 nucleotides.

9. The shRNA polynucleotide of claim 8 , wherein the first nucleotide of the polynucleotide overhang is double stranded.

10. The shRNA polynucleotide of claim 8 , wherein the first nucleotide of the polynucleotide overhang is single stranded.

11. The shRNA polynucleotide of claim 8 , wherein the second nucleotide of the polynucleotide overhang is single stranded.

12. The shRNA polynucleotide of claim 8 , wherein the third nucleotide of the polynucleotide overhang is single stranded.

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