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Fig. 11
EGFP-based in vivo assay for the elucidation of miRNA/siRNA pathway activators. Cells expressing
EGFP were treated with shRNA targeting the gene and an array of small molecules. Enoxacin was found to
increase the degree of EGFP silencing relative to a non-treated control, signifying an activator of the RNAi
pathway. Image adapted with permission from Shan G et al. (2008) Nat Biotechnol
2,000 FDA-approved compounds,
14
was found to enhance
siRNA-mediated degradation of mRNA as GFP silencing was
higher than the cellular controls (Fig.
11
). Interestingly, the Xi
laboratory also developed a similar assay and independently discov-
ered the same compound as a positive effector [
60
].
Though other structurally similar molecules were also
screened, enoxacin was the most effective at enhancing RNAi
activity. Microarray analyses demonstrated that enoxacin-treated
cells did not display signifi cant changes in gene expression, indi-
cating that enoxacin likely affects shRNA/pre-miRNA matura-
tion. Northern blot analysis showed that the presence of enoxacin
increased the levels of both pre-let-7 and pre-miR-30 only in the
presence of both Dicer and TRBP (but not Dicer alone). This
trend suggests that enoxacin likely aids in the loading of RNA
