Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 36

Fig. 2.2 “R” form duplexes are more potent than “L” form duplexes. ( a ) An EGFP expression
plasmid was transfected into HEK293 cells. Anti-EGFP or control dsRNAs were transfected 24 h
later at the indicated concentrations. At 24 h post-transfection, relative fluorescence was measured
setting the control cultures at 100%. ( b ) The EGFP site 1 mRNA target sequence is shown at the
top with 21-nt and asymmetric 25/27-nt DsiRNAs aligned below. RNA bases are uppercase and
DNA bases are lowercase . “p” indicates a 5¢-phosphate. Reprinted from Rose et al. [ 31 ] with per-
mission from Oxford University Press

ejected as the passenger strand. The primary feature contributing to loading bias
relates to thermodynamic end asymmetry [ 33- 35 ]; it now appears that end structure
(3¢-overhang vs. blunt) also plays a role [ 31, 32 ]. Interestingly, a recent report from
the Doudna group demonstrated that Dicer is integrally involved in strand selection
during RISC formation, lending additional credibility to the functional polarity
observed using Dicer-substrate siRNAs in the studies discussed above [ 36 ] .

2.2.1.3 Functional Potency of DsiRNAs

A study was performed at the Bio-Rad laboratories by Eli Hefner, Teresa Rubio, and
colleagues to validate the performance of the newly optimized DsiRNA design [ 37 ] .
A set of five genes were selected as targets, including ACTB , AKT1 , RAF1 , CDK2 ,
and TP53 . For each gene, an asymmetric 25/27-nt DsiRNA targeting that gene and
the cognate 21-nt siRNA were compared for potency and duration of silencing.