Methods and compositions relating to polypeptides with RNase III domains that mediate RNA interference
First Claim
1. A method of reducing expression of a target gene in a cell comprising:
- a) incubating a dsRNA corresponding to part of the target gene with an effective amount of a composition comprising a polypeptide comprising an RNase III domain, under conditions to allow RNase III to cleave the dsRNA into siRNA; and
b) transfecting the siRNA into the cell.
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Abstract
The present invention concerns methods and compositions involving RNase III and polypeptides containing RNase III domains to generate RNA capable of triggering RNA-mediated interference (RNAi) in a cell. In some embodiments, the RNase III is from a prokaryote. RNase III activity will cleave a double-stranded RNA molecule into short RNA molecules that may trigger or mediate RNAi (siRNA). Compositions of the invention include kits that include an RNase III domain-containing polypeptide. The present invention further concerns methods using polypeptides with RNase III activity for generating RNA molecules that effect RNAi, including the generation of a number of RNA molecules to the same target.
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Citations
37 Claims
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1. A method of reducing expression of a target gene in a cell comprising:
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a) incubating a dsRNA corresponding to part of the target gene with an effective amount of a composition comprising a polypeptide comprising an RNase III domain, under conditions to allow RNase III to cleave the dsRNA into siRNA; and
b) transfecting the siRNA into the cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for achieving RNA interference of a target gene in a cell using one or more siRNA molecules comprising:
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a) generating at least one double-stranded DNA template corresponding to part of the target gene, wherein the DNA template comprises an SP6, T3, or T7 promoter on at least one strand;
b) transcribing the template, wherein either i) a single RNA strand with a complementarity region, or ii) first and second complementary RNA strands is/are created;
c) hybridizing either the single complementary RNA strand or first and second complementary RNA strands to create a dsRNA molecule corresponding to the target gene;
d) incubating the dsRNA molecule with a polypeptide comprising an RNase III domain, under conditions to allow cleavage of the dsRNA into at least two siRNA; and
e) transfecting at least one siRNA into the cell. - View Dependent Claims (11, 12, 13)
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14. A kit for generating siRNA molecules comprising:
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a) recombinant, prokaryotic RNase III;
b) RNase III buffer; and
c) a control nucleic acid. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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- 29. A method for generating siRNA that can reduce expression of a target gene comprising incubating a dsRNA corresponding to part of the target gene with an effective amount of a composition comprising a polypeptide comprising an RNase III domain, under conditions to allow RNase III to cleave the dsRNA into siRNA.
Specification