Synthetic shRNAs as potent RNAi triggers.
Designing potent silencing triggers is key to the successful application of RNA interference (RNAi) in mammals. Recent studies suggest that the assembly of RNAi effector complexes is coupled to Dicer cleavage. Here we examine whether transfection of optimized Dicer substrates results in an improved RNAi response. Dicer cleavage of chemically synthesized short hairpin RNAs (shRNAs) with 29-base-pair stems and 2-nucleotide 3' overhangs produced predictable homogeneous small RNAs comprising the 22 bases at the 3' end of the stem. Consequently, direct comparisons of synthetic small interfering RNAs and shRNAs that yield the same small RNA became possible. We found synthetic 29-mer shRNAs to be more potent inducers of RNAi than small interfering RNAs. Maximal inhibition of target genes was achieved at lower concentrations and silencing at 24 h was often greater. These studies provide the basis for an improved approach to triggering experimental silencing via the RNAi pathway.
Siolas
Despina
D
Lerner
Cara
C
Burchard
Julja
J
Ge
Wei
W
Linsley
Peter S
PS
Paddison
Patrick J
PJ
Hannon
Gregory J
GJ
Cleary
Michele A
MA
D005786:Gene Expression Regulation / Q000235:genetics*; D020868:Gene Silencing / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D034741:RNA, Small Interfering