Synthetic Biology Parts, Devices and Applications

170 8 Design of Ligand‐Controlled Genetic Switches Based on RNA Interference

(triggers) and control RNAi‐mediated gene expression (referred to as RNAi

switches). To obtain such RNAi switches, appropriate RNA sequences located in

(pri)(pre)‐miRNAs or shRNAs have been engineered to modulate the recogni-

tion of RNA‐processing nucleases such as Drosha and Dicer, making it possible

to bind to various triggers and inhibit or permit nuclease processing. To design

the trigger molecule‐controlled RNAi switches, it is useful to isolate functional

RNA modules based on RNA secondary structures because RNA is often divided

into functional modules and reassembled through the double‐stranded regions

without disrupting the original function. Synthetic RNAi switches have been

developed by employing various trigger molecules (e.g., small molecules, RNA,

or proteins) that take advantage of the modularity of RNA.

8.2 Development of RNAi Switches that Respond

to Trigger Molecules

Control of gene expression from exogenous DNA by a set of transcription factors

and coupled small molecules has conventionally been used for conditional

expression strategies [6–8]. Similarly, the transcriptional control of shRNA

expression using small molecules has been employed for the construction of tun-

able genetic switches based on RNAi [9]. This system has also combined the Lac

inhibitor with shRNA RNAi to synergistically suppress target gene transcription

and translation.

Drosha

(processing)

Pre-miRNA

Exportin-5

(nuclear exporting)

Nucleus

Cytoplasm

Dicer

(processing)

Mature miRNA

Tr ansf

ection

Pri-miRNA

shRNA transcription

from plasmidDNA

RISC (Ago2)

(Target mRNA degradation)

siRNA

shRNA

Cell membrane

(m)RNA transcription from plasmid orgenomic DNA Ta rget gene repression

siRNA

Figure 8.1 Schematic of the RNAi pathway in mammalian cells, including endogenous miRNA

processing and ectopic shRNA or siRNA expression.