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Scheme 2 Synthesis of aminoglycoside dimers via “click” chemistry
assay and found to have IC 50 values between 0.5 and 20
μ
M
(relative to 50-100
M exhibited by kanamycin). These results
suggest that aminoglycosides represent useful privileged core
structures for the inhibition of miRNA maturation. However, the
specifi city of these molecules has yet to be investigated, and increas-
ing their affi nity for general RNA binding may lead to issues within
a cellular environment.
These aminoglycoside inhibitors were then employed in the
development of a more robust assay for the measurement of pre-
miRNA maturation based on branched rolling-circle-amplifi cation
(BRCA) [ 56 ]. Though a variety of assays have been developed to
monitor the maturation of miRNA, especially in the presence of
inhibitors, current methods contain many functional limitations.
Previously described [ 32 ] P-labeled pre-miRNA PAGE assays to
evaluate miRNA maturation inhibition are tedious and unsuitable
for high-throughput screenings. Comparatively, BRCA allows for
sensitive quantifi cations of miRNA levels. This particular method
utilizes a cyclic DNA template with a complementary primer that
through polymerase binding triggers the formation of single-
stranded DNA product. The single-stranded DNA then serves as a
template for further amplifi cation with a secondary primer. Using
this technique, Arenz et al. designed a label-free BRCA-based assay
that could selectively detect the formation of mature miRNAs.
In order to engineer discrimination between pre-miRNA and
mature miRNA, the circular DNA template was constructed so
that only mature miRNA could initiate BRCA. The 3
μ
end of
mature miRNA, which is inaccessible in the pre-miRNA, was used
for BRCA elongation (Fig. 9 ). After designing a 65-mer circular
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