Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 32

experiments performed in the laboratories of John Rossi at the Beckman Research
Institute of the City of Hope National Medical Center and at Integrated DNA
Technologies [ 30, 31 ] .

2.2.1 Characterization of Synthetic Dicer-Substrate siRNAs

2.2.1.1 Duplex Length and Structure

A series of blunt-ended RNA oligonucleotide duplexes were tested for cleavage in
an in vitro dicing assay [ 30 ]. The synthetic dsRNAs were incubated with recombi-
nant human Dicer, desalted, and subjected to electrospray ionization (ESI) mass
spectrometry. Duplexes as short as 23-nt length were cleaved to 21-nt length, and
duplexes from 23- to 30-nt length all showed efficient cleavage. Cleavage efficiency
decreased as length was extended to 35, 40, and 45 nts. Thus, synthetic RNA
duplexes can function as Dicer substrates, and the optimal length for in vitro dicing
was estimated to be in the 25-30-nt range.
The effect of duplex end structure was investigated, and Dicer cleavage occurred
whether the duplex had blunt ends, 5¢-overhangs, or 3¢-overhangs; however, func-
tional potency varied significantly with structure (see below). The effect of end
modification (i.e., the addition of non-nucleotide moieties) was investigated by
placing a bulky fluorescein group at the 5¢ -end, 3 ¢-end, or both ends of each strand
of the duplex. 5¢-modification was well tolerated, but dicing efficiency was mark-
edly reduced by the introduction of a single 3¢ -modi fi cation on either end of the
duplex. Cleavage was entirely blocked if the duplex was modified at both 3¢ -ends.
Functional potency in gene knockdown correlated with dicing efficiency. These
observations are consistent with a mechanism where the Dicer PAZ domain first
binds the substrate RNA at the 3¢-end and then cleavage follows; any modification
of structure that interferes with 3¢-end binding in these very short substrate RNAs
disrupts processing.

2.2.1.2 Optimized Design of Dicer-Substrate siRNAs

Functional potency of a series of anti- EGFP RNA duplexes was tested by transfec-
tion into EGFP-expressing HEK297 cells [ 30 ]. The length of the RNA duplexes was
varied from 21 to 30 nts, having either 5¢ -overhangs, 3 ¢-overhangs, or blunt ends
[ 30 ]. All of the duplexes tested showed effective suppression of EGFP fluorescence
when used at a high concentration (50 nM), but only the longer duplexes showed
efficacy when the concentration used was reduced to subnanomolar levels (50-
200 pM). At this site, a 27-nt blunt duplex was the most potent compound tested,
and the EC 50 shifted from 20 nM for the 21-nt siRNA to 200 pM for the 27-nt blunt
duplex. A prolonged duration of silencing was also observed for the 27-nt duplex,
with detectable EGFP suppression increasing from 4 to 10 days; the increased dura-
tion of silencing may simply reflect the higher potency of the compound. Significant