Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 30

Chapter 2
Synthetic Dicer-Substrate siRNAs
as Triggers of RNA Interference

Scott D. Rose and Mark A. Behlke

Abstract The first synthetic oligonucleotides used to suppress gene expression in
mammalian cells via RNA interference were 21-nucleotide (nt) RNA duplexes hav-
ing symmetric 2-nt 3¢-overhangs and were designed to mimic the natural products
of Dicer processing of long RNA substrates. Synthetic RNA duplexes which are
longer than 23-nt length are substrates for processing by Dicer and can show
increased potency as artificial triggers of RNA interference, particularly at a low
concentration. Longer duplexes, however, can have variable cleavage patterns fol-
lowing Dicer processing which can adversely affect potency. Optimized synthetic
Dicer substrates are asymmetric duplexes having a 25-nt passenger strand and a
27-nt guide strand with a single 2-nt 3¢-overhang on the guide strand and modified
bases at the 3¢-end of the passenger strand. This modified design results in predict-
able patterns of Dicer processing and shows improved activity. The development of
this design strategy and use of Dicer-substrate RNAs to trigger gene suppression in
a variety of systems will be reviewed in this chapter.

2.1 Introduction

RNA interference (RNAi) is a highly conserved mechanism of gene regulation that
extends broadly across phyla [ 1 ]. RNAi encompasses two general mechanisms of
gene suppression, one where the target mRNA is degraded and a second where pro-
tein translation is inhibited [ 2, 3 ]. Both routes reduce levels of the protein product
made from the targeted gene. Translational suppression is typically mediated by
microRNAs (miRNAs), which form imperfect hybrids with the target mRNA [ 4 ] .

S. D. Rose, PhD • M. A. Behlke , MD, PhD ( * )
Integrated DNA Technologies, Inc. , 1710 Commercial Park , Coralville , IA , USA
e-mail: [e-mail]