Rna interference agent, method for producing same, and use therefor
AssigneeGifu University
InventorsYukio KITADE, Remi NAKASHIMA
Life of this patent
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Abstract
Provided is an RNA interference agent, with which a suppression effect on the off-target effect can be obtained with a simple system. The disclosures relate to an RNA interference agent provided with a single-stranded oligonucleotide passenger strand having one or two or more PAZ domain low-affinity units at the 3′-end.
Claims17
Suppressing agent of off-target effect comprising a single-stranded oligonucleotide provided with one or two or more units at the 3′-end, wherein each unit is represented by the following Formula (1):
- [Chem. 16]
- [—X—Y—O—]n Formula(1)
- (where X is an acidic group or basic group or a linking group having these, Y represents a group with 2 or 3 carbon atoms, and n is an integer of 2 or more).
An RNA interference agent, which is provided with:
- a guide strand oligonucleotide having an antisense site for a specific nucleotide sequence of a target gene and one or two or more PAZ domain high-affinity units at the 3′-end;
- and
- a passenger strand oligonucleotide having one or two or more PAZ domain low-affinity units at the 3′-end and hybridizes specifically with the antisense site,
- wherein the PAZ domain high-affinity unit has an equal to or higher affinity than the affinity that TT strand has to PAZ domain, and
- the PAZ domain low-affinity unit has an lower affinity than that TT strand has to PAZ domain.
A method for producing an RNA interference agent, the method comprising:
- forming a double-stranded RNA by hybridizing specifically a guide strand oligonucleotide having an antisense site for a specific nucleotide sequence of a target gene and one or two or more PAZ domain high-affinity units at the 3′-end and a passenger strand oligonucleotide having one or two or more PAZ domain low-affinity units at the 3′-end and hybridizes specifically with the antisense site.
An synthetic reagent for off-target suppressing agent represented by the following formula:
- W1O—Y—OW2 [Chem. 20]
- (where Y represents a alkylene group with 2 or 3 carbons, W1 represents a hydrogen atom or hydroxy protecting group, and W2 is a hydrogen atom or phosphoramidite group or a linking group that binds to or has been bound to a solid-phase carrier).
A method of screening an RNA interference agent, the method comprising:
- preparing one or two or more RNA interference agents each having a guide strand with an antisense site for a specific nucleotide sequence in a target gene and a passenger strand with one or two or more candidate PAZ domain low-affinity units at the 3′-end;
- and
- introducing the one or two or more RNA interference agents into a cell carrying the target gene and evaluating changes in the cell.
Dependencies are inferred from the claim text.