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Therefore, small molecules may represent a suitable alternative to
miRNA regulation, as they are less expensive to produce, readily
diffuse across cell membranes, are easily delivered into cells, ani-
mals, and humans with high temporal resolution, and are more
stable intracellularly. Lastly, small molecules have more ideal drug
properties including good solubility, good bioavailability, and met-
abolic stability [ 10 ].
Small molecules have the potential to control miRNA expres-
sion on multiple levels of the miRNA pathway such as pre-
transcription, transcription, and posttranscription, in contrast to
nucleic acid-based reagents that can only regulate miRNA func-
tion through a direct interaction with the mature miRNA.
Although all three levels of regulation are important, both pre-
and posttranscriptional regulation appear to be generally less
miRNA-specifi c, whereas the transcriptional level offers a higher
degree of miRNA specifi city since transcription factors are pre-
sumably involved in the development- and cell-specifi c regulation
of distinct miRNAs [ 12 ]. Consequently, small molecules are more
versatile probes to study miRNA biogenesis than nucleic acid-
based tools.
In recent years, several small molecule screens have been con-
ducted in order to identify compounds that either activate or
deactivate miRNAs [ 13 - 19 ]. Most small molecules that have
been identifi ed regulate miRNA expression in a general fashion
by either inhibiting or activating miRNA processing. Our
approach to small molecule regulation of miRNAs focuses on the
discovery and development of small molecules that inhibit spe-
cifi c miRNAs in order to use the identifi ed small molecules as
molecular probes for those particular miRNAs of interest. We
have developed assay systems to discover miRNA-specifi c inhibi-
tors and have identifi ed several small molecules that could be use-
ful tools for the elucidation of detailed mechanisms of miRNA
action and may serve as lead structures for the development of
new therapeutic agents. Specifi cally, we have focused on the miR-
NAs miR-21 and miR-122.
MiRNA miR-122 is required by the hepatitis C virus (HCV)
for replication and infectious virus production [ 20 ]. It was recently
reported that knockdown of miR-122 with antisense agents
resulted in a decrease in HCV RNA replication in human liver cells
[ 20 ] and reduced HCV levels in chronically infected primates [ 21 ],
suggesting that small molecule inhibitors that target miR-122
could be a viable avenue for a fundamentally new antiviral
therapy.
A reporter assay for mature miR-122 function was constructed
based on the psiCHECK-2 (Promega) reporter plasmid [ 14 ]. The
complementary sequence for mature miR-122 was inserted down-
stream of the Renilla luciferase gene to create the reporter
construct psiCHECK-miR122, and a stable Huh7 reporter cell
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