Methods and compositions for the specific inhibition of gene expression by double-stranded RNA
First Claim
1. A precursor RNAi molecule capable of reducing the expression of a target gene following cleavage by Dicer comprising:
- a first oligonucleotide strand that is 22-30 nucleotides in length; and
a second oligonucleotide strand that is 22-30 nucleotides in length and comprises a nucleotide sequence that is sufficiently complementary to a sequence of an RNA of the target gene to direct target-specific RNAi and wherein the second oligonucleotide strand anneals to the first oligonucleotide strand under biological conditions, wherein at least one of said first and second oligonucleotide strands is modified on the 3′
end to direct Dicer processing of the precursor RNAi molecule and wherein said precursor RNAi is a substrate for Dicer.
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Abstract
The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. The dsRNA has a first oligonucleotide sequence that is between 25 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of at least 19 nucleotides is sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger the destruction of the target RNA by the RNAi machinery.
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Citations
110 Claims
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1. A precursor RNAi molecule capable of reducing the expression of a target gene following cleavage by Dicer comprising:
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a first oligonucleotide strand that is 22-30 nucleotides in length; and
a second oligonucleotide strand that is 22-30 nucleotides in length and comprises a nucleotide sequence that is sufficiently complementary to a sequence of an RNA of the target gene to direct target-specific RNAi and wherein the second oligonucleotide strand anneals to the first oligonucleotide strand under biological conditions, wherein at least one of said first and second oligonucleotide strands is modified on the 3′
end to direct Dicer processing of the precursor RNAi molecule and wherein said precursor RNAi is a substrate for Dicer. - 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, 34, 35, 36, 37, 38, 75, 76, 77, 78, 79, 80, 81, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 108)
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39. A precursor RNAi molecule capable of reducing the expression of a target gene following cleavage by Dicer comprising:
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a first oligonucleotide strand that is 22-30 nucleotides in length; and
a second oligonucleotide strand that is 22-30 nucleotides in length and comprises a nucleotide sequence that is sufficiently complementary to a sequence of an RNA of the target gene to direct target-specific RNAi and wherein the second oligonucleotide strand anneals to the first oligonucleotide strand under biological conditions, wherein the first and second oligonucleotide strands have a different length and wherein one of said first and second oligonucleotide strands has a 3′
overhang that enhances Dicer processing of the precursor RNAi molecule and wherein said precursor RNAi is a substrate for Dicer. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 82, 83, 84, 85, 86, 109)
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62. A precursor RNAi molecule capable of reducing the expression of a target gene following cleavage by Dicer comprising:
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a first oligonucleotide strand that is 25-30 nucleotides in length; and
a second oligonucleotide strand that is 25-30 nucleotides in length and comprises a nucleotide sequence that is sufficiently complementary to a sequence of an RNA of the target gene to direct target-specific RNAi and wherein the second oligonucleotide strand anneals to the first oligonucleotide strand under biological conditions, wherein said precursor RNAi is a substrate for Dicer. - View Dependent Claims (63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 87, 104, 105, 106, 107, 110)
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Specification