Biology Reference
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Fig.
1
Homogenous assay of miRNA maturation. Native structure of pre-miRNA beacon is cleaved by Dicer
enzyme. Cleavage cuts off quencher and fl uorophore leading to fl uorescent increase. In case of potent inhibi-
tors, Dicer cleavage is blocked and quenching is still on
Monitoring the Dicer-mediated miRNA maturation step by
this homogenous assay based on a fl uorescent substrate has great
potential: (1) Any desired pre-miRNA can be labeled with
fl uorophore and quencher to validate miRNA maturation using
recombinant Dicer enzyme or cell lysate. (2) Assay can be used to
determine Dicer activity of cell lysate with respect to one specifi c
miRNA. (3) Potential inhibitors of miRNA maturation can be
tested in the manner of high-throughput screen using recombi-
nant Dicer enzyme [
6
,
7
]. The homogenous assay of miRNA mat-
uration (Fig.
1
) is based on a fl uorescent pre-miRNA probe that
contains a 5
-quencher (Q). Before cleav-
age, fl uorescence is quenched because of the close proximity of
fl uorophore and quencher in the native hairpin formation of pre-
miRNA. Dicer cleaves that native structure and enables the fl uoro-
phore to emit its fl uorescence without quenching, which can be
detected at the proper wavelength. In the presence of an inhibitor
that binds to pre-miRNA, Dicer cleavage is blocked and quenching
persists. Thus, a lower or no increase in fl uorescence is observed.
Especially for screening purposes this assay can be used in 384-well
microplates using a plate reader (
see
Note 2
).
It should be mentioned that Dicer usually recognizes its sub-
strates via interaction with the specifi c 5
′
-fl uorophore (F) and a 3
′
- two
nucleotide overhang at the terminus of the pre-miRNA stem [
8
].
Cleavage specifi city then is most probably set by the spatial dis-
tance of the dsRNA from the specifi c recognition site. The lack of
this specifi c signature may lead to a shifted cleavage of pre-miRNA
[
9
], which cannot be excluded for end-labeled pre-miRNA as
described here. Such a shift in the cleavage site is irrelevant for the
evaluation of relative Dicer activity, but may affect the validity of
results, when pre-miRNA binders as potential inhibitors of miRNA
maturation are screened. However, by directly comparing results
for such inhibitor testings using this assay and an alternative assay
relying on unmodifi ed pre-miRNA, we found no signifi cant differ-
ences [
10
]. Recently, similar assays for Dicer activity [
11
] or
miRNA maturation [
12
] have been described.
′
-phosphate and 3
′
