Synthetic Biology Parts, Devices and Applications

8.5 Future Perspectives 177

bloodstream, efficient uptake by tumors, and reassociation‐triggered activa-
tion of split siRNA functioning [18]. MON‐triggered pre‐miRNA switches can
be applied to reporters of activated siRNA molecules in individual cells. Equal
amounts of MON (including siRNA and pre‐miRNA targeting for fluorescent
proteins) are produced in the nucleus when Drosha cleaves MON‐bound pre‐
miRNA switches [15]. The levels of RNAi and siRNA molecules can thus be
more precisely monitored and visualized than with co‐transfection of target
and reference (fluorescent protein target) siRNA. Protein‐triggered shRNA
switches can be applied to control cell fate. Protein‐triggered shRNA switches
can respond to human U1A and NFκB protein expression within cells [26].
L7Ae‐triggered shRNA switches were shown to control human cell fate by reg-
ulating the balance between proapoptotic (Bim) and antiapoptotic (Bcl‐xL)
protein molecules via the knockdown of antiapoptotic proteins (Bcl‐xL) and by
determining the status of mitochondrial‐dependent apoptosis pathways. The
expression of L7Ae determines cell survival.

8.5 Future Perspectives

Recent intensive research has resulted in the development of RNAi switches that
are triggered by multiple chemicals and biomacromolecules. To improve the
ability of RNAi switches to rewire gene regulatory networks, however, there
are  several challenges to overcome regarding switch efficiency and the variety
of  specific trigger molecules for other RNA switches. For example, protein‐
triggered shRNA switches require high plasmid expression levels of the trigger
protein. To generate switches that respond to endogenous protein molecules,
designed RNA devices must efficiently and selectively detect target proteins [28].
Extra signal amplification systems such as synthetic positive feedback loops may
be required to generate sufficient protein signals. Additionally, an orthogonal
RNA–protein‐binding pair that does not interfere with natural RNA or protein
molecules is desirable to sense target proteins without inducing side effects.
Thus, it is important to develop an automated and easy selection method to
generate such specific RNA–protein‐binding pairs from RNA motif libraries.

Exogenous trigger

Small
molecules

DNA/MON

Endogenous trigger

RNA Proteins

RNAi ON/OFF switches

RNAi

OFF

Reporter genes ON

Antiapoptotic

gene

Proapoptotic

gene

Reporter genes OFF

Cell death

RNAiON Cell survival

Figure 8.4 Applications of RNAi switches. RNAi ON/OFF switches can be applied to RNAi
reporter and cell fate conversion systems that respond to specific trigger molecules.