174 8 Design of Ligand‐Controlled Genetic Switches Based on RNA Interference
inhibitory sequence, resulting in perfect hybridization of the sense and antisense
strands. The resulting dsRNA is processed as active siRNA by Dicer (OFF‐to‐ON
RNAi switch). MON‐triggered pri‐miRNA switches were also designed by intro-
ducing an inhibitory RNA stem loop into the 3′ end of the pri‐miRNA to conceal
the single‐stranded region of the Drosha recognition site [15]. MON targets
endogenous genes, and the pri‐miRNA contains a target sequence for a fluores-
cent protein (EGFP or DsRed) in EGFP‐ or DsRed‐expressing HeLa cells. When
present, MON perfectly hybridizes with half of the inhibitory RNA stem‐loop
sequence, resulting in an RNA conformational change and exposure of the
single‐ stranded region that is recognized by Drosha. The resulting dsRNA is also
processed as an siRNA by Dicer.8.2.3 Protein‐Triggered RNAi Switches
Protein‐triggered RNA switches have been designed by replacing the loop region
of shRNA with protein‐binding sequences in an attempt to mask the Dicer rec-
ognition site in the presence of trigger protein molecules [16, 17]. The specific
and tight RNA–protein interaction (RNP) motif is important when designing an
efficient RNAi switch. For these switches, an RNP motif consisting of an archaeal
ribosomal protein, L7Ae, and its binding partner, box C/D kink‐turn RNA (Kt),
is employed to develop L7Ae‐triggered shRNA switches (Kt‐shRNA) [16]. L7Ae
binds to the loop region of Kt‐shRNA, which inhibits Dicer cleavage and targets
gene knockdown. Following Kt‐shRNA development, designed shRNA switches
triggered by the human splicing‐related protein U1A and the human transcrip-
tional regulator NFκB (p50 domain) were developed [17]. The RNP motifs of the
U1A protein and the loop sequence of U1 snRNA or the loop‐stem‐loop sequence
in the 3′ untranslated region of U1A mRNA were utilized to develop two types of
U1A‐triggered shRNA switches. An RNP motif composed of the NFκB protein
and an artificially selected NFκB‐binding aptamer was also employed to develop
an NFκB‐triggered shRNA switch. The molecular structures of these RNP motifs
were solved via crystal or nuclear magnetic resonance (NMR) structural analyses
and utilized to create three‐dimensional (3D) molecular designs of the switches.
The switches were designed by incorporating a protein‐binding sequence into
the loop region of shRNA, which contains 22–28 bp of dsRNA targeting the
EGFP gene in the stem region. The configurations of these switches were three‐
dimensionally optimized such that the interaction between the trigger protein
and shRNA efficiently blocked Dicer processing.8.3 Rational Design of Functional RNAi Switches
Rational and predictable RNA design strategies are critical for developing versa-
tile RNAi switch systems. Hereafter, we will focus on design strategies for RNAi
switches. The most common design strategy for RNA switches utilizes predicted
RNA secondary structures and their free energies based on Watson–Crick base
pairing in the presence/absence of trigger molecules. This strategy also attempts
to optimize the free energy difference between the two states by changing base