Biology Reference
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EGFP) fused to an RNA aptamer evolved for the small molecule
theophylline. A control construct was also prepared which lacked
the aptamer motif on the shRNA. Monitoring of the GFP fl uores-
cence was correlated to the ability of the shRNA to silence gene
expression. The addition of theophylline led to the dose-dependent
increase in fl uorescence for the shRNA fused to the RNA aptamer;
however, the theophylline had no effect on the control shRNA.
RNAi inhibition by theophylline recovered the EGFP signal, and a
similar result was also obtained when targeting the DsRed gene.
A variety of constructs were developed in order to elucidate
theophylline's mechanism of action. The results indicated that the-
ophylline's interaction with the RNA aptamer inhibits Dicer
cleavage likely due to the close proximity of the binding pocket to
the Dicer cleavage site. To observe the inhibition of siRNA in vivo,
researchers co-transfected an EGFP and an shRNA expression vec-
tor and then incubated cells in the presence or the absence of
10 mM theophylline. Northern blot analysis revealed that siRNA
production was almost completely inhibited when the RNA
aptamer and theophylline were both present. Conversely, there was
no signifi cant change in the siRNA levels for the vector lacking the
theophylline binding aptamer. Though multiple aptamer con-
structs were prepared, the highest degree of inhibition was
observed when the putative Dicer cleavage site overlapped with the
theophylline-binding site.
Though multiple methods have aimed to use small molecules
as a means to regulate RNAi pathways, they involve careful design
and engineering. The aptamer-fused shRNA system may be used in
combination with other regulatory systems that also target the
RNAi pathway. The benefi t of this particular aptamer-fused
approach is a faster, more direct response than with transcriptional
control achieved by targeting the Dicer-mediated cleavage.
However, to be more applicable in the targeting of miRNAs, this
methodology must be adapted to produce shRNAs targeting miR-
NAs directly, and other approaches previously described appear to
be somewhat more straightforward. Yet, one signifi cant advantage
is the ability to modulate miRNA function at a given time via intro-
duction of the small-molecule effector.
5
Conclusions
The ever-increasing relevance of miRNAs within biological systems
requires the development of new tools to better understand and
modulate its function. Specifi cally, the ability to modulate its matu-
ration using either small molecule or biomacromolecule approaches
has a plethora of applications including the development of novel
therapeutics for multiple diseases and disorders. While RNA has
traditionally been considered a “non-druggable” target many
