8.4 Application of the RNAi Switches 175pair lengths and introducing mutations [11, 15, 22–25]. Several ligand‐ (e.g.,
small molecule or oligonucleotide) responsive RNAi switches have been designed
based on this strategy. When the secondary structure, free energy difference, and
base pair length have been optimized, a similar design strategy could be applied
to generate various RNAi switches that respond to different trigger molecules.
A useful and efficient 3D design approach has been utilized to develop protein‐
triggered RNAi switches by employing available 3D RNP structures (analyzed via
both NMR and X‐ray crystallography) [17, 26]. For the first approach, the struc-
tural components of shRNA switches were three‐dimensionally reconstructed in
silico by creating 22–28 bp of A‐form dsRNA with 3D molecular design software
and loading the RNP motif, composed of the trigger protein and its binding RNA
motif, from the Protein Data Bank (Figure 8.3a, left). Then, 3D structural models
of the trigger protein‐bound shRNA switches were constructed by superimpos-
ing the few terminal nucleotides of the RNA loop on the dsRNA using minimiza-
tion methods consisting of the least squares approximation polynomial and
connecting the loop with the dsRNA. The models predicted the structural states
of the shRNA switches in the presence of the trigger protein (Figure 8.3a, right).
As described in Figure 8.3, the bound trigger protein on the shRNA switch
rotates approximately 30° in a counterclockwise direction around the axis of the
dsRNA with a 1‐bp insertion and is located ~2.6 Å farther from the site of Dicer
cleavage.
Because the Dicer enzyme can access the 22nd nucleotides from both the
5 ′ and 3′ ends [27], the bound trigger protein on the switches was designed to
block Dicer access. Specifically, the base pair lengths of the switches were
adjusted by taking advantage of the orientation change of each base pair such
that the bound trigger proteins could block Dicer access (Figure 8.3b). To predict
in silico the collision between Dicer and the bound trigger protein, the con-
structed switch models were superimposed on the catalytic sites and the periph-
eral region of Giardia Dicer with reference to the Dicer cleavage sites and
catalytic sites. Based on the results of the 3D molecular design and switch assess-
ment, steric hindrance between Dicer and the shRNA‐bound protein was pre-
dicted in silico, which positively correlated with the inhibition of Dicer cleavage
in vitro and target gene expression in living cells. Furthermore, the 3D molecular
design method could be applied for all switches that sense several different RNA‐
binding proteins (e.g., L7Ae, U1A, and NFκB) and could be used to predict the
functions of these proteins. In principle, the strategy could predict functional
switch structures in response to RNA‐binding proteins to adjust the ON/OFF
ratio of the designed switches.
8.4 Application of the RNAi Switches
RNAi switches have been proposed for applications including drug delivery,
RNAi reporters, conditional knockdown, and cell fate controls (Figure 8.4). For
example, DNA‐mediated siRNA switches consist of DNA–RNA hybrids that
may be suitable for the systemic delivery of siRNA. In vivo (mouse) studies
have demonstrated that these switches promote degradation resistance in the