Biology Reference
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[
11
]. This method involves radioactive labeling of RNA and
tedious polyacrylamide gel electrophoresis, making it unsuitable
for high-throughput screening. Qiagen offers a quantitative PCR
kit for the separate quantifi cation of miRNA and pre-miRNA from
one sample [
12
], and researchers from Pfi zer have published an
assay to evaluate Dicer cleavage effi ciency of double-stranded RNA
[
13
]. The fi rst in vitro assay that directly monitors Dicer-meditated
miRNA maturation has been developed in our laboratory and uses
fl uorecently labeled pre-miRNAs. Therein, the 5
′
-end of the pre-
miRNA is attached to a fl uorophore and the 3
-end to a quencher,
which leads to absorption of the emitted fl uorescence presumably
through collisional quenching when the typical hairpin structure is
formed. Incubation with Dicer produces small double-stranded
miRNAs and leads to ineffective quenching and increase in fl uores-
cence, and this can then be used to monitor the Dicer cleavage
process [
14
,
15
]. However, as mentioned above, it is not clear
how these pre-miRNA modifi cations infl uence the Dicer cleavage.
It has been reported that modifi cations to the 5
′
-ends can
cause a shift in the cleavage site and, hence, a modifi ed mature
miRNA sequence, and that the effi ciency and specifi city of the
cleavage through Dicer are infl uenced [
10
]. Therefore, modifi ca-
tions of the pre-miRNA for a miRNA maturation assay should be
avoided. Though reporter gene assays exist and have been used to
screen small-molecule libraries for the inhibition of miRNA matu-
ration, the exact molecular target of thus-identifi ed inhibitors
remains unclear and has to be examined in further experiments
[
16
,
17
]. The group of Maiti has circumvented this limitation by
screening a number of amingoglycosides—known as RNA bind-
ers—with such a reporter gene assay [
18
]. The inhibition of pre-
miR-21 maturation was afterwards confi rmed by a maturation
assay using
32
P-labeled RNA. An in vitro assay allowing the detec-
tion of solely the Dicer-mediated cleavage of pre-miRNA to mature
miRNA would allow an immediate evaluation of test compounds.
Jonstrup et al. [
19
] and Cheng et al. [
20
] applied the known
isothermal branched rolling circle amplifi cation (BRCA) technique
to quantify miRNAs in cell lysate. To enhance specifi city in their
quantifi cation method, they use a ligase for the miRNA-templated
circularization of the ssDNA padlock probe. However, both groups
did not clarify the issue of selectivity between pre-miRNA and
mature miRNA. We have modifi ed this detection method towards
a rapid functional in vitro assay to detect miRNA maturation using
unmodifi ed pre-miRNA. Therein, a designed circular ssDNA (
see
Note 1
) carries a hybridization site for the mature miRNA (Fig.
1
).
The miRNA that originates from the 5
′
- and 3
′
-arm of the pre-miRNA
then serves as a primer for isothermal amplifi cation with
Bacillus
stearothermophilus
(Bst) DNA poylmerase (large fragment,
see
Note 2
). The usage of a secondary primer complementary to the
generated concatamer enhances amplifi cation. Unlike the mature
miRNA, the pre-miRNA cannot serve as a primer, since the 3
′
′
-OH
