Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 34

knockdown at two different sites in the STAT1 gene are compared between a 21-nt
siRNA and two blunt 27-nt dsRNAs that overlap the 21-nt sequence. At site 1, one
of the two 27-nt blunt dsRNAs showed low potency, while the other showed high
potency, similar to a 21-nt siRNA at this site. At site 2, both of the 27-nt dsRNAs
showed lower potency than the 21-nt siRNA.
To investigate these seemingly inconsistent results, an assay was developed at
Integrated DNA Technologies to study the products of Dicer processing of the syn-
thetic RNA duplexes, hoping to find some correlation between Dicer processing
patterns and functional potency. The synthetic dsRNAs were incubated with recom-
binant human Dicer, desalted, and subjected to electrospray ionization (ESI) mass
spectrometry. Using these methods, single Dalton accuracy in mass measurement
was achieved, which, in most cases, permitted precise identification of the species
produced by Dicer cleavage [ 31 ]. It was found that blunt 27-nt substrate dsRNAs
were usually cleaved by Dicer into several products. Sometimes as many as five or
more different species were observed, which varied in size from 20 to 23 nts.
Furthermore, the precise pattern of cleavage could not be readily predicted from
sequence. Significant differences in potency are sometimes seen between siRNAs
that are shifted by as little as a single base along a target sequence; thus, small dif-
ferences in dicing patterns could account for large differences in observed potency.
These observations also imply that a direct comparison of potency between a blunt
27-nt dsRNA and the “cognate” 21-nt siRNA is really only possible if the dominant
21-nt siRNA produced by Dicer cleavage is identified by a mass spectrometry assay
(or other means). Without this information, there is no way to know which of the
possible 21-nt siRNAs that could potentially be cleaved from a 27-nt dsRNA sub-
strate is the correct species to employ in a comparative study.
In summary, the mass spectrometry and functional studies discussed above indi-
cated that it was not possible to predict the potency of blunt 27-nt synthetic
dsRNAs; sometimes Dicer cleavage resulted in highly potent 21-nt siRNAs from a
substrate, and other times low potency species were produced. To improve on these
results, a thorough structure-activity relationship (SAR) study was performed where
duplexes of different length and design were studied for cleavage patterns and func-
tional potency with the goal of finding a dsRNA substrate that gave predictable
results with Dicer processing.
The Dicer-substrate SAR studies identified a design that showed particular
promise [ 31 ]. The lead compound was an asymmetric dsRNA with a 25-nt sense
(passenger) strand and 27-nt antisense (guide) strand. This design has a single
3¢-overhang which resides on the guide strand and is blunt at the other end; in addi-
tion, two DNA bases were placed at the 3¢-end of the passenger strand. Dicer pro-
cessing of this substrate usually resulted in production of a single 21-nt siRNA
species which spanned from the original 3¢-overhang in the substrate to a cleavage
point 21 bases away. Henceforth, this asymmetric 25/27-nt RNA duplex will be
called a “DsiRNA” (optimized Dicer-substrate siRNA). While it is not certain why
this design was so effective at encouraging uniform processing, the authors specu-
lated that the single 3¢-overhang was bound by the Dicer PAZ domain which posi-
tions the substrate at a specific distance from the active endonuclease site, thereby
resulting in more uniform processing than was seen using blunt substrates where