Biology Reference
In-Depth Information
Fig. 10
Quadruplex binding compounds found to inhibit maturation of shRNA
aminoglycosides may afford a heightened degree of specifi city and
inhibition of certain pre-miRNAs.
As a means of achieving some form of specifi city of pre-miRNA
processing, work in the Hartig laboratory utilized known
G-quadruplex small-molecule binders [
58
]. While this study was
conducted using short hairpin RNAs (shRNAs) (an analog of pre-
miRNAs in the siRNA pathway), specifi c G-rich RNA sequences
were able to bind bisquinolinium (
12
) and porphyrazine (
13
)
compounds with 20-400 nM dissociation constants and inhibit
Dicer processing as demonstrated via SDS-PAGE analysis (Fig.
10
).
While this method is not amenable to high-throughput screening
and some knowledge of the pre-miRNA secondary is required, it
does represent a means of obtaining some specifi city for G-rich
sequences in pre-miRNAs. Unfortunately, when translated to a
mammalian cell system the presence of the small molecules was not
able to reverse the silencing effects of the shRNAs on a reporter
gene. This is potentially the result of poor cellular uptake of the
compounds or inability to discriminate between the desired shR-
NAs and various other G-quadruplexes present within the cell.
Thus, in order to develop practical compounds, a signifi cantly
higher degree of specifi city must be achieved for the targeted
pre-miRNA.
3.4 Modulation of
Pre-miRNA In Vivo
Non-specifi cally
Recently, some classes of small molecules have also been implicated
in the upregulation of RNA interference pathways by increasing
Dicer-mediated processing. By developing a cell-based screening
assay, Shan et al. identifi ed the small molecule enoxacin (
14
) as a
promoter of miRNA biogenesis [
59
]. Researchers developed an
assay in HEK293T cells that stably expressed both a GFP reporter
and an shRNA. The shRNA required maturation by Dicer to yield
a siRNA that specifi cally targets the GFP mRNA, resulting in a
reduction of fl uorescence. Using this reporter system to screen
