Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 37

Fig. 2.3 Functional polarity is introduced by Dicer processing. Two forms of DsiRNAs are shown:
the “L” form duplex with the single 3¢-overhang on the sense strand (S) and the “R” form duplex
with the single 3¢-overhang on the antisense strand (AS). Sites of Dicer cleavage are indicated by
a gap in the sequence; R = RNA, D = DNA. A schematic image of Dicer showing the different
functional domains is overlaid on the dsRNA substrate, positioning the RNase III domains at the
staggered cut sites and the PAZ domain at the single 3¢-overhang. The strand of the cleavage prod-
uct which is favored for loading into RISC as the “guide strand” is highlighted in bold , demonstrat-
ing how functional polarity could be introduced by differential positioning of the antisense strand
in Dicer between the “L” and “R” forms
Each RNA duplex was transfected into HeLa cells using the siLentFect™ cationic
lipid reagent at concentrations of 50 nM, 5 nM, 1 nM, and 100 pM. RNA was
extracted 24 h post-transfection, and RT-qPCR was performed to assess relative
knockdown of the target mRNA. In four of the five genes studied ( ACTB , RAF1 ,
CDK2 , and TP53 ), the DsiRNA showed higher potency than the cognate 21-nt
siRNA, especially at the lower doses. For the other target ( AKT1 ), the DsiRNA and
siRNA showed identical potency. The dose response results for the anti- TP53
DsiRNA and siRNA are shown in Fig. 2.4a , and the sequences employed are shown
in Fig. 2.4c .
A comparison of the duration of silencing was also performed. The DsiRNA and
siRNA pairs were individually transfected into HeLa cells at 5 nM concentration,
and cultures were sampled at days 1, 2, 4, and 6 post-transfection. RNA was
extracted, and RT-qPCR was performed to assess relative knockdown of the target
mRNA. The results paralleled the dose-response data discussed previously, and, for
four of the five genes studied ( ACTB , RAF1 , CDK2 , and TP53 ), the DsiRNAs
showed longer duration of silencing than their cognate siRNAs. For the other target