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pre-miRNA
inactive small molecule
small molecule inhibitor
Dicer cleavage
Dicer inhibition
fluorescence
fluorescence quenching
Fig. 8 Pre-miRNA maturation assay for Dicer processing. A dual-labeled pre-miRNA with a fl uorescent mole-
cule and a quencher in close proximity is incubated with Dicer in vitro. Small-molecule inhibition of processing
prevents RNA cleavage and maintains fl uorescence quenching, while inactive molecules result in Dicer pro-
cessing and a fl uorescent signal
disruption of pre-miRNA processing is most likely nonspecifi c; how-
ever, by screening against large-peptide or small-molecule libraries
with different pre-miRNA reporters it may be feasible to identify
specifi c inhibitors of the processing event. Either way, the targeting
of Dicer-mediated maturation represents an excellent target in
decreasing the intracellular concentrations of mature miRNAs.
Based on the well-documented ability of aminoglycosides to target
and bind RNA, they become a logical source for pre-miRNA bind-
ers to modulate maturation. As noted in the previous assay kana-
mycin was able to elicit some effect on Dicer processing of pre-let-7
miRNA, suggesting the viability of this class of compounds as
inhibitors. Towards this end a series of aminoglycoside derivatives
based on 2-deoxystreptamine ( 11 ) and neamine ( 10 ) derivatives
were prepared and dimerized via 1,3-dipolar cycloaddition reac-
tions to increase the valency and thus potency of their RNA bind-
ing activity (Scheme 2 ) [ 55 ]. Six conjugates of 10 and 11 were
assayed in the previously described fl uorescence quenching Dicer
3.3.2 Inhibition of
Pre-miRNA Maturation
by Aminoglycosides
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