Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 31

Degradative RNAi, on the other hand, is typically mediated by small interfering
RNAs (siRNAs), which form perfect or near-perfect hybrids with the target mRNA
[ 5 ]. There is substantial “cross talk” between the pathways, and miRNAs can lead
to mRNA degradation, and siRNAs can lead to translational suppression [ 6 ] .
Endogenous siRNAs are symmetric 21-nt RNA duplexes having a 19-nt duplex
domain and 2-nt 3¢-overhangs that are processed from longer double-stranded (ds)
RNAs by the endoribonuclease Dicer [ 7- 9 ]. Dicer functions as a heterodimer with
a second RNA-binding protein, R2D2 in Drosophila or TRBP (the human
immunodeficiency virus transactivating response RNA-binding protein) in humans
[ 10- 12 ]. Dicer is a large protein with multiple functional domains, including two
RNase H family nuclease domains, which perform substrate cleavage, and a PAZ
domain, which binds short single-stranded RNA overhangs [ 13, 14 ] . Binding of an
RNA overhang by the PAZ domain orients the substrate within Dicer. The first
nuclease domain is separated from the PAZ domain by a “connector helix,” which
determines the distance between the PAZ binding site and the cleavage site, which
is 21-22 bases for mammalian Dicer enzymes. Following cleavage, the nascent
siRNA remains associated with Dicer and TRBP. An Argonaute (Ago) family pro-
tein then associates with the complex, forming a functional RNA-induced silencing
complex (RISC) [ 15, 16 ]. The siRNA is transferred from Dicer/TRBP to the Ago
protein, where the siRNA is converted to single-stranded (ss) form by either cleav-
age/degradation of one strand or unwinding by a helicase activity [ 17- 19 ] . The
ejected or degraded strand is called “the passenger strand,” and the retained strand
is called “the guide strand.” The guide strand directs the sequence specificity of all
subsequent gene suppression activity of the complex. Like Dicer, the Ago proteins
possess a PAZ domain which binds the 3¢-single-stranded overhang of the siRNA
and orientates it within the complex [ 20 ]. There are four Argonaute proteins in
humans that perform different effector functions in RISC [ 21- 24 ] . In particular,
Ago2 is an endoribonuclease which cleaves the target mRNA as directed by sequence
complementarity to the siRNA guide strand bound in RISC [ 25- 27 ] and is the key
protein responsible for degradative RNAi.
The first generation of chemically synthesized siRNAs was designed to mimic
the natural products of Dicer, i.e., 21-nt RNA duplexes with 2-nt 3¢ -overhangs [ 28 ] .
This design remains the dominant form of synthetic siRNAs in use today. During
the 10 years since this initial discovery, a variety of artificial designs have been
proposed to improve upon some aspect of the RNAi process, which were discussed
in a review by Chang and colleagues [ 29 ]. This chapter will review development
and use of Dicer-substrate siRNAs (DsiRNAs) as a trigger of RNAi.

2.2 Development of Dicer-Substrate siRNA Technology

Dicer is involved in RISC loading, so it is possible that using a synthetic RNA
duplex that is a substrate for Dicer and thus engages Dicer prior to RISC assembly
may show different properties as a trigger for RNAi rather than an RNA duplex that
mimics a Dicer product. This hypothesis was tested in a series of collaborative