Method and compositions for RNA interference
OwnerMichaeline Bunting, Knut Madden +1inventors
InventorsMichaeline Bunting, Knut Madden, Adam Harris
Life of this patent
Estimating the term…
Abstract
The invention provides methods and compositions related to the field of gene expression regulation. In particular, methods and compositions of the invention can be used to identify RNAi cleavage sites along a target RNA molecule. Methods and compositions of the invention may also be used to knockdown expression of nucleic acid molecules which encode reporters.
Claims51
A method for identifying one or more RNAi cleavage sites along a target RNA molecule, the method comprising:
- introducing one or more double stranded RNA (dsRNA) molecules into one or more cells comprising the target RNA molecule, wherein the nucleotide sequence of at least one of the strands of the one or more dsRNA molecules is identical to a nucleotide sequence found within the target RNA molecule;
- incubating the one or more cells under conditions which allow for cleavage of the target RNA molecule, thereby producing two or more target RNA fragments;
- releasing RNA from the cells;
- determining the nucleotide sequence of (i) one or more of the target RNA fragments, or (ii) one or more terminal portions of one or more of the target RNA fragments;
- and
- comparing the sequence data obtained in (d) to the sequence of the target RNA molecule.
A method for identifying one or more RNAi cleavage sites along a target RNA molecule, the method comprising:
- introducing a mixed population of double stranded RNA (dsRNA) molecules into one or more cells comprising the target RNA molecule, wherein the nucleotide sequence of at least one of the strands of each member of the mixed population of dsRNA molecules is identical to a nucleotide sequence found within the target RNA molecule;
- incubating the one or more cells under conditions which allow for cleavage of the target RNA molecule, thereby producing two or more target RNA fragments;
- releasing RNA from the cells;
- determining the nucleotide sequence of (i) one or more of the target RNA fragments, or (ii) one or more terminal portions of one or more of the target RNA fragments;
- and
- comparing the sequence data obtained in (d) to the sequence of the intact target RNA molecule.
(canceled)
(canceled)
A method for producing a mixed population of double stranded RNA (dsRNA) fragments, the method comprising:
- incubating a first intact dsRNA molecule with an enzyme having RNase activity, thereby producing a first set of two or more dsRNA fragments;
- incubating a second intact dsRNA molecule with an enzyme having RNase activity, thereby producing a second set of two or more dsRNA fragments;
- and
- combining the first set of two or more dsRNA fragments with the second set of two or more dsRNA fragments, thereby producing a mixed population of dsRNA fragments;
- wherein the first intact dsRNA molecule and the second intact dsRNA molecule are non-identical.
A method for producing a mixed population of double stranded RNA (dsRNA) fragments, the method comprising:
- combining a first intact dsRNA molecule and a second intact dsRNA molecule to form a mixture of intact dsRNA molecules;
- incubating the mixture of intact dsRNA molecules with an enzyme having RNase activity, thereby producing a mixed population of dsRNA fragments;
- wherein the first intact dsRNA molecule and the second intact dsRNA molecule are non-identical.
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
A mixed population of double stranded RNA (dsRNA) molecules, the mixed population comprising at least one first dsRNA molecule and at least one second dsRNA molecule,
- wherein the nucleotide sequence of at least one of the strands of the first dsRNA molecule is at least 90% identical to the nucleotide sequence encoded by a first gene or a portion thereof,
- wherein the nucleotide sequence of at least one of the strands of the second dsRNA molecule is at least 90% identical to the nucleotide sequence encoded by a second gene or a portion thereof, and
- wherein the first and the second dsRNA molecules are non-identical.
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
An isolated dsRNA molecule comprising a nucleotide sequence, at least one strand of which is identical to at least 10 nucleotides of a messenger RNA which encodes a polypeptide with β-lactamase activity.
(canceled)
(canceled)
Dependencies are inferred from the claim text.