Biology Reference
In-Depth Information
the ability to be performed with whole-cell lysates or recombinant
Dicer. Furthermore, the BRCA-based assay provides a useful means
of monitoring miRNA maturation, without the use of expensive
and time-consuming labeling.
Aminoglycosides have also been examined in another assay
involving fl uorescent intercalator displacement (FID) to assess
their binding and inhibition of the processing of miR-151 [
57
].
This particular miRNA was selected as it has been shown to have a
pivotal role in immune function and its high expression has been
shown to cause cancer in mice. In this assay ethidium (RNA K
d
5
M) was prebound to the pre-miRNA target, followed by incu-
bation with an aminoglycoside. A decrease in fl uorescence signal
could then be correlated to the ability of the aminoglycoside to
displace the intercalated ethidium molecule. Of the ten
aminoglycosides screened, fi ve demonstrated moderate affi nity to
the RNA target in the low micromolar range. To confi rm actual
binding of the molecules, rather than a simple structural alterna-
tion of the RNA target, direct binding studies were conducted via
probing the thermal melting temperature of the pre-miRNA tar-
get. An increase in melting temperature corresponds to a binding
interaction between the RNA and the aminoglycoside, leading to
increased stabilization of the complex. The data suggests that ami-
noglycosides in particular are effective in binding to the pre-miRNA
target and do so in a dose-response relationship with ligand
concentration.
When examined in a Dicer-catalyzed pre-miRNA processing
SDS-PAGE assay, an interesting effect was observed, as only three
of the aminoglycosides exhibited any inhibitory effect at all (~10 %)
despite binding the pre-miRNA. Additionally, the assay demon-
strated that degradation products corresponding to nonspecifi c
Dicer processing were observed and were also inhibited by the
aminoglycosides. This indicates that the aminoglycosides are not
only nonspecifi c inhibitors but also nonspecifi c binders. This pro-
vides a cautionary point that demonstrates that RNA binding is
not necessarily correlated to the inhibition of pre-miRNA matura-
tion. Consequently, a higher degree of specifi city and inhibitory
activity is a requisite before any of these compounds can have ther-
apeutic relevance.
μ
In the previously described FID assay by Herdewijn et al., other
small-molecule RNA binders were examined, most notably various
oligonucleotide intercalators [
57
]. Many of these molecules,
including DAPI, profl avine, doxorubicin, and Hoechst 33258,
exhibited high levels of both binding and inhibition by SDS-PAGE
analysis of Dicer-mediated pre-miRNA processing. However, these
compounds were found to be neither specifi c binders nor specifi c
inhibitors, as nonspecifi c Dicer cuts were also inhibited. It is specu-
lated that potential hybrids of these intercalating compounds with
3.3.3 Inhibition of
Pre-miRNA Maturation
by Other Small Molecules
