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Publication Number
US-8758998-B2
Publication Date
2014-06-24
Expiration Date
Abstract
A method for designing a bi-shRNA expression cassette encoding a bi-shRNA comprising: selecting one or more target site sequences; providing a backbone sequence comprising a first and a second stem-loop structure, inserting a first passenger strand and a second passenger strand and providing for synthesis of the bi-shRNA expression cassette.
Core Innovation
The disclosed invention relates to a method for designing a bi-shRNA expression cassette encoding a bifunctional short hairpin RNA (bi-shRNA). The bi-shRNA comprises a backbone sequence with a first and a second stem-loop structure, wherein the first and second stem-loop structures are linked by a sequence longer than 5 nucleotides. Each stem-loop structure provides two insertion sites linked by a loop sequence within a corresponding stem.
The method includes selecting one or more first target site sequences and one or more second target site sequences of a targeted species. The selected first target site sequences have low homology with other mRNAs of the targeted species, where low homology is selected from specified thresholds of less than 75%, less than 80%, less than 90%, less than 95%, or less than 98% homology. Passenger and guide strands are inserted into the insertion sites to form the first stem-loop structure and the second stem-loop structure with defined complementarity and partial complementarity rules.
For the second stem-loop structure, the passenger and guide strands are designed to be partially complementary, where partially complementary is defined as having 1 or 1-2, or 1-3, or 1-4, or 1-5, or 1-6, or 1-7, or 1-8, or 1-9, or 1-10 nucleotide mismatches when paired. In dependent refinements, the method further includes determining the ΔG free energy of the second stem-loop structure and, if beyond about −10 Kcal·mole−1, introducing additional mismatches at the 3′ half of the second passenger strand, and specifying mismatch locations at particular nucleotide positions.
Claims Coverage
The document provides one independent claim describing a design method for a bi-shRNA expression cassette, with multiple dependent claims that narrow target-site selection, structural constraints, and mismatch/thermodynamic constraints. Across the independent claim, the inventive coverage centers on three inventive features.
Low-homology target-site selection for first and second targets
Selecting one or more first target site sequences and one or more second target site sequences of a targeted species, wherein the one or more first target site sequences have low homology with other mRNAs of the targeted species, where low homology is selected from the group consisting of less than 75%, less than 80%, less than 90%, less than 95%, and less than 98% homology.
Backbone with two linked stem-loop structures
Providing a backbone sequence comprising a first and a second stem-loop structure, wherein the first stem-loop structure comprises two first insertion sites linked by a first loop sequence within the first stem and wherein the second stem-loop structure comprises two second insertion sites linked by a second loop sequence within the second stem, wherein the first and the second stem-loop structures are linked by a sequence longer than 5 nucleotides.
Partially complementary passenger/guide insertion to form the second stem-loop structure
Inserting a second passenger strand and a second guide strand into the two second insertion sites to form the second stem-loop structure, wherein either the second passenger strand or the second guide strand is homologous with the one or more first target site sequences, or wherein either the second passenger strand or the second guide strand is homologous with the one or more second target sites sequences, wherein the second passenger strand and the second guide strand are partially complementary, wherein partially complementary is defined has having 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 nucleotide mismatches when paired.
Overall, the claim coverage is grounded in designing a bi-shRNA expression cassette with two linked stem-loop structures whose passenger/guide strand content is determined by low-homology target-site sequence selection and by partially complementary mismatch-defined pairing.
Stated Advantages
Documented Applications
No documented applications found
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