Synthetic Biology Parts, Devices and Applications

8.2 Development of RNAi Switches that Respond to  rigger Molecules 171

Hereafter, we will focus on an RNA design strategy and the posttranscriptional
gene expression control of RNAi switches via several trigger molecules including
small molecules, oligonucleotides, and proteins (Figure 8.2). These triggers bind
to specific RNA sequences, and the interactions between them can be employed
to generate RNAi switches (Table 8.1). The advantages and potential applications
of RNAi switches primarily depend on the type of trigger. Small molecule trig-
gers that penetrate through the cell membrane tune the function of RNAi
switches by adjusting the extracellular concentration of the input molecules,
which is a mechanism similar to that of small molecule‐inducible transcription
factors. Oligonucleotide triggers, such as DNA, RNA, and modified oligonucleo-
tides (MONs), are able to form Watson–Crick base pairs with designed RNA
devices and thus adjust specificities and affinities between the trigger molecules
and the devices. Protein triggers can also be used to control the functions of
RNAi switches. Thus, specific proteins expressed in cells can distinguish target
cell types based on the intracellular environment.

8.2.1 Small Molecule‐Triggered RNAi Switches

Small molecule‐triggered RNAi switches have been designed to modulate Dicer
or Drosha processing of shRNA or pri‐miRNA. Initially, three different switch
design strategies implementing a theophylline aptamer were employed to achieve
theophylline‐responsive properties [20]. In the first design approach to obtain
theophylline‐responsive shRNA switches, the loop region of EGFP‐ (or DsRed‐)
targeting shRNA was replaced with a theophylline aptamer containing a loop
sequence; this replacement was designed to create a theophylline and RNA com-
plex around the Dicer recognition site [10]. When expressed in HEK293 cells, the
switches inhibited Dicer processing and knockdown of reporter fluorescent
genes in the presence of theophylline in culture medium. A similar approach was
applied to the development of pri‐miRNA‐based RNAi switches (pri‐miRNA

Trigger molecules

Pri-miRNA

Pre-miRNA

shRNA

siRNA

RNAi inducing RNA sequence

Small molecules

Oligo nucleotides

Proteins

RNA motif

Specific

binding

Design and optimization for functional switches

Inplant

RNAi switches

Figure 8.2 RNAi switch design strategies with a variety of trigger molecules. The RNA motifs
that bind to specific trigger molecules are introduced into the appropriate regions in
pri‐miRNA, pre‐miRNA, shRNA, or siRNA. The motifs embedded in the RNA are then optimized
to generate functional RNAi switches.