SELECTING AND STABILIZING dsRNA CONSTRUCTS
First Claim
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1. A method of obtaining a nucleic acid segment providing a desired level of suppression of a target gene, comprising:
- a) obtaining a starting nucleic acid molecule substantially complementary to a target gene;
b) preparing a plurality of nucleic acid segments from the starting nucleic acid molecule;
c) assaying the nucleic acid segments for the ability to suppress expression of the target gene when expressed as a dsRNA in a cell comprising the target gene; and
d) identifying at least a first nucleic acid segment from the plurality of nucleic acid segments that provides a desired level of suppression of the target gene when expressed as a dsRNA.
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Abstract
The invention provides methods for selecting nucleotide sequences that yield dsRNA-mediated gene suppression in a target organism and enable their uptake by the target organism. The invention further provides expression constructs that confer stabilized expression of such sequences in a transgenic host cell, and methods for their use. Also provided are organisms, cells and tissues prepared by a method of the invention.
69 Citations
98 Claims
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1. A method of obtaining a nucleic acid segment providing a desired level of suppression of a target gene, comprising:
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a) obtaining a starting nucleic acid molecule substantially complementary to a target gene;
b) preparing a plurality of nucleic acid segments from the starting nucleic acid molecule;
c) assaying the nucleic acid segments for the ability to suppress expression of the target gene when expressed as a dsRNA in a cell comprising the target gene; and
d) identifying at least a first nucleic acid segment from the plurality of nucleic acid segments that provides a desired level of suppression of the target gene when expressed as a dsRNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 96, 97, 98)
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34. A method of producing an expression construct for expressing a dsRNA with reduced transgene silencing in a plant cell, comprising:
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a) preparing an expression construct comprising a first sequence, a second sequence, and a third polynucleotide sequence, wherein the third polynucleotide sequence is linked to the first polynucleotide sequence by the second polynucleotide sequence and the third polynucleotide sequence is substantially the reverse complement of the first polynucleotide sequence; and
b) introducing an intron into at least one of the first and third polynucleotide sequences or introducing said expression construct into the intron, wherein the first and third polynucleotide sequences hybridize when transcribed into RNA and form a dsRNA molecule stabilized by the second polynucleotide sequence after intron splicing, and wherein the expression construct exhibits reduced transgene silencing in a plant cell transformed with the expression construct relative to an expression construct that lacks the intron. - View Dependent Claims (35, 36, 37, 38, 39, 40)
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41. A method of increasing the pest or pathogen-inhibitory activity of a dsRNA, comprising
a) obtaining a first nucleic acid segment that when expressed as a dsRNA and taken up by a target crop pest or pathogen inhibits feeding by the target crop pest or pathogen or progeny thereof; - and
b) linking the first nucleic acid segment to a second nucleic acid segment to create a longer nucleic acid segment, wherein the second nucleic acid segment is a nucleic acid that does not inhibit feeding by the target crop pest or pathogen or progeny thereof when expressed as a dsRNA, and wherein a dsRNA expressed from the longer nucleic acid exhibits increased potency of inhibition of feeding by the target crop pest or pathogen or progeny thereof relative to the dsRNA expressed from the first nucleic acid segment alone. - View Dependent Claims (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63)
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64. A method of producing an expression construct for expressing a dsRNA with increased specificity of pest or pathogen-inhibitory activity comprising:
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a) obtaining a starting nucleic acid molecule substantially complementary to at least a first coding sequence of a target crop pest or pathogen;
b) selecting a region within the starting nucleic acid molecule that when expressed as a dsRNA inhibits feeding by the target crop pest or pathogen or progeny thereof following uptake of the dsRNA expressed from the region by the target crop pest or pathogen;
c) linking the region to a second nucleic acid molecule to produce an expression construct, wherein the second nucleic acid molecule when expressed as a dsRNA does not inhibit feeding by a target crop pest or pathogen or progeny thereof following uptake of the dsRNA. - View Dependent Claims (65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)
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- 83. A method of enhancing the control of a target crop pest or pathogen in a plant comprising expressing in the cells of the plant at least two dsRNA sequences that function upon uptake by the pest or pathogen to inhibit the expression of at least a first target coding sequence within the target crop pest or pathogen, wherein the two dsRNA sequences are substantially complementary to two non-contiguous portions of the first target coding sequence or to two different coding sequences of the target crop pest or pathogen.
Specification