Design and Experimental Validation of Small Activating RNAs Targeting an Exogenous Promoter in Human Cells

It
is increasingly practical to co-opt many native cellular components
into use as elements of synthetic biological systems. We present the
design and experimental investigation of the first exogenous genetic
construct to be successfully targeted by RNA activation, a phenomenon
whereby small double-stranded RNAs increase gene expression from sequence-similar
promoters by a mechanism thought to be related to that of RNA interference.
Our selection of activating RNA candidates was informed by a custom-written
computer program designed to choose target sites in the promoter of
interest according to a set of empirical optimality criteria drawn
from prior research. Activating RNA candidates were assessed for activity
against two exogenously derived target promoters, with successful
candidates being subjected to further rounds of validation as a precaution
against potential off-target effects. A genetic platform was assembled
that allowed activating RNA candidates to be simultaneously screened
both for positive activity on the target reporter gene and for possible
nonspecific effects on cell metabolism. Several candidate sequences
were tested to appraise the utility of this platform, with the most
successful achieving a moderate activation level with minimal off-target
effects.