Synthetic shRNAs as potent RNAi triggers.
Nat Biotechnol. 2005 Feb;23(2):227-31

LDespina Siolas¹, ², Cara Lerner³, Julja Burchard³, Wei Ge³, Peter S Linsley³, Patrick J Paddison², Gregory J Hannon² & Michele A Cleary<sup>3

1</sup> Program in Genetics, Stony Brook University, Stony Brook, New York 11794, USA.
² Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
³ Rosetta Inpharmatics, LLC, a wholly owned subsidiary of Merck and Co., Inc., 401 Terry North, Seattle, Washington 98109, USA.

Abstract

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.