Biology Reference
In-Depth Information
Fig. 9
Branched rolling-circle-amplifi cation (BRCA) assay for detection of pre-
miRNA maturation inhibitors. If the pre-miRNA is not fully processed, the residual
hairpin blocks polymerase amplifi cation of the circular DNA template, whereas if
pre-miRNA is fully matured exponential amplifi cation of the template can occur.
Image adapted with permission from Neubacher S et al. (2011) Chem Bio Chem
DNA template with a sequence complementary to mature let-7
miRNA, real-time PCR was used to monitor the BRCA reactions.
When two polymerases, Kle
-exo
and Bst, were used in the presence
of the control, untreated pre-let-7, BRCA reactions were not
observed. Conversely, when the pre-let-7 template was preincu-
bated with recombinant Dicer, there was signifi cant DNA polym-
erization. The Dicer cleavage and BRCA reaction could be
conducted in an effi cient one-pot reaction due to the reaction opti-
mal temperatures for Kle
-exo
and Dicer. However, due to the 40-fold
increased signal amplifi cation Bst was selected to optimize the
reaction despite requiring a higher temperature. To measure the
selectivity for precursor or mature miRNA, pre-let-7 was incubated
with Dicer for increasing periods of time. The results indicated a
strong dependence on Dicer incubation time, making this assay
particularly useful for measuring miRNA maturation. Moreover,
the assay can also selectively monitor miRNA maturation in the
presence of multiple miRNAs.
The BRCA-based assay was further tested to determine the
effects of the known aminoglycoside inhibitors of Dicer cleavage.
A control BRCA reaction was designed in order to exclude poten-
tial false hits that could occur if the inhibitor altered polymeriza-
tion patterns. The results of these assays yielded similar inhibition
values as determined by the previously described fl uorescence-
based assays, confi rming its validity as a screen. However, this
method provides several benefi ts over other techniques, including
