8.2 Development of RNAi Switches that Respond to rigger Molecules 173switches), and the theophylline aptamer was introduced into the Drosha
recognition site of pri‐miRNA [14]. The second strategy to design switches was
based on the changing stability of the two RNA reversible conformational states
(active or inactive) via trigger (theophylline or hypoxanthine) binding [11]. In the
absence of triggers, shRNA switched from the canonical dsRNA structure (active
state) that is required for EGFP knockdown; the binding of the trigger changed
the secondary structure of the switches, and part of one dsRNA strand stably
bound to the adjacent loop sequence (inactive state) and collapsed the canoni-
cal dsRNA structure. The third strategy employed an irreversible conforma-
tional change of the pri‐miRNA structure and ligand‐controlled hammerhead
ribozymes [12]. In the absence of theophylline, the allosteric hammerhead
ribozyme domain and the following inhibitory strand that hybridizes the pri‐
miRNA collapsed the canonical structure that is required for Drosha processing.
In the presence of theophylline, ribozyme self‐cleavage induced the exposure of
the 5′‐single‐stranded region that was originally masked by the inhibitory strand,
resulting in Drosha processing. Another design strategy considered endogenous
pre‐miRNA as potential RNAi switches. In this system, Dicer or Drosha process-
ing was inhibited by bioactive small molecules that target pre‐miRNAs [19]. The
strategy employed the tight binding pair of a benzimidazole and RNA internal
loop motif from a database of RNA motif‐small molecule interactions and
searched the Dicer or Drosha recognition sites of disease‐related pre‐miRNA for
the RNA internal loop motif [19]. The motif was found and well fit with the
Drosha recognition site of human pre‐miR‐96. In the result the benzimidazole
specifically inhibited the endogenous pre‐miR‐96 maturation, recovered the
downstream proapoptotic gene FOXO1 expression and induced apoptosis in
MCF7 cells.
8.2.2 Oligonucleotide‐Triggered RNAi Switches
Oligonucleotide‐triggered RNAi switches have been designed to modulate Dicer
processing of siRNA or Drosha processing of pri‐miRNA. The strategy for
designing the switches is based on toehold‐mediated oligonucleotide displace-
ment. DNA‐mediated siRNA switches are composed of two DNA–RNA hybrids
[18]. The RNA strands of the hybrids are split sense and antisense strands
of siRNA. The DNA strands contain additional nucleotides (toeholds) in the
hybridization region, and the two DNA strands can potentially form a double
strand. The initial DNA–RNA hybrid is not processed by Dicer because it cannot
recognize a DNA–RNA hybrid [21]. After the DNA–RNA hybrids are trans-
fected into mammalian MDA‐MB231 cells, the DNAs bind to each other at the
toehold region and replace RNA with antisense DNA to produce double‐stranded
DNA and siRNA. The siRNA is then processed by Dicer to knock down target
genes.
A small RNA‐triggered siRNA switch was designed to block the double‐
stranded formation of sense strand and antisense strand RNA in the absence of a
trigger. For this, an inhibitory sequence is connected to the 3′ end of the sense
strand and is partially hybridized with the antisense strand‐binding region of the
sense strand [13]. Meanwhile, the trigger RNA when present hybridizes with an