Synthetic shRNAs as potent RNAi triggers

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.

Grass carp reovirus activates RNAi pathway in rare minnow, Gobiocypris rarus

“…Long (27-29 bp) dsRNAs and short hairpin RNAs (shRNAs) that enter the RNAi pathway in a Dicer-dependent fashion provide more efficient gene silencing than shorter Dicer-independent substrates (Kim et al, 2004;Siolas et al, 2005). Recent studies have also suggested that dsRNAs greater than 23 bp can influence cell viability and induce a potent IFN response (highlighted by a strong up-regulation of the dsRNA receptor, Toll-like receptor 3) in a cell type-specific manner.…”

Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes

“…8 hairpins stems can be more efficient triggers of down-regulation than the 19 nt stems generated using the pSUPER RNAi System™ (Siolas et al, 2005); for optimal efficiency it would therefore be important to test a selection of oligonucleotides for different organisms.…”

Synthetic circuits, devices and modules

“…Furthermore, the elucidation of functional roles of small interfering RNAs and microRNAs has identified them as potent gene post-transcriptional regulators. Due to their effectiveness in gene silencing in an array of organisms, small synthetic interfering RNAs (Kim et al, 2005;Siolas et al, 2005;Beisel et al, 2008) and microRNAs (Dickins et al, 2005;Suryawanshi et al, 2010) have been used to control gene expression.…”