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investigated this hypothesis by constructing various decoys, termed
microRNA sponges, that contain tandem miRNA-binding sites.
These transcripts were inserted into the 3
UTR of a destabilized
GFP gene (used to measure transformation effi ciency) driven by
either a CMV promoter or a U6 small nuclear RNA promoter-ter-
minator vector. The RNA sequence of the sponges was then modi-
fi ed to be either perfectly or imperfectly complementary to the
miRNA of interest.
Expression assays with
Renilla
luciferase genes regulated by
miR-20 revealed an increase in relative target expression after co-
transfection with either CMV or U6 sponges. Both types of
sponges inhibited miR-20, rescuing the expression of the luciferase
target, with the imperfectly matched sponges outperforming the
perfectly complementary sponges. The ability of miRNA sponges
to inhibit endogenous miRNA was compared to previously devel-
oped antisense therapies by transfecting with target reporters.
While most of the oligonucleotides depressed the target reporter
expression level, the most signifi cant change was observed with the
miRNA sponges (Fig.
13
). The combined use of other oligonucle-
otides and miRNA sponges resulted in the largest recovery of target
expression levels.
Another benefi t of miRNA sponges is the ability of a single
sponge to inhibit miRNA families that share a common heptameric
seed. A target reporter with binding sites for miR-30c was used to
verify the seed specifi city of a miRNA sponge against miR-30e
compared to a 2
′
-O-methyl antisense oligonucleotide against miR-
30e. The sequences for miR-30e and miR-30c have the same hep-
tameric seed; however the antisense oligonucleotide only slightly
depressed the target miR-30c. The miR-30e sponge restored target
expression over fourfold, indicating cross-reactivity with multiple
miR-30 family members.
While antisense oligonucleotides provide an effi cient means to
inhibit endogenous miRNAs, miRNA sponges act as effi cient
decoys that can target multiple miRNA family members. Future
work with miRNA sponges aims at increasing their potency, per-
haps through additional seed-binding sites and modulation of their
complementarity.
Due to the gene regulatory role of miRNAs, especially in devel-
opmental processes, the immediate knockdown of miRNA function
upon introduction of an antisense agent may not be optimal and
result in undesired side effects. Towards this end the Li laboratory
developed photoactivatable antisense oligonucleotides that remain
inactive in their miRNA silencing until a brief irradiation with
365 nm light [
63
]. This affords a degree of spatial and temporal
control over the antisense reagent, allowing for complex investiga-
tions into the role of miRNAs in developmental processes.
Specifi cally, a blocking sequence complementary to the antisense
reagent was intramolecularly attached to the 2
′
′
-O-methyl antisense
