Biology Reference
In-Depth Information
Fig. 5
Functional assay for the screening of immobilized peptoid miRNA binders. Fluorescently labeled miRNAs
and RNA controls are incubated with the peptoid library and washed, and peptoids with a high affi nity retain
the labeled RNA. Binding can be assessed by fl uorescence microscopy to detect the labeled RNA. Specifi city
of the binding can be assessed by comparing plates incubated with the miRNA to the plates incubated with
RNA control
different RNA targets. While the strategy readily lends itself to the
rapid synthesis of diverse libraries, the requisite for immobilization
to a solid-support becomes somewhat limiting. Moreover, while
peptoid binding will most likely inhibit pre-miRNA processing, it
has yet to be demonstrated in an in vivo or an in vitro context.
However, future work probing the specifi city of this diverse library
against other pre-miRNAs and even pri-miRNAs may yield highly
stable compounds that can readily be employed towards the inhibi-
tion of miRNA maturation.
3.2 Specifi c Binding
of Pre-miRNA by
Peptides and Peptoids
In Vivo
Though many research approaches aim to inhibit miRNA function
through the use of oligonucleotides or peptides to obtain target
specifi city in vivo, recent work has strived to employ small molecules
to impede miRNA maturation. Such techniques provide several
advantages over antagomir and other peptide methods, including
more effective delivery to tissues. One such class of small mole-
cules, known as aminoglycosides, is known to bind to secondary
structures within RNA, such as stem-loops. Since miRNA precur-
sors contain stem-loops as well as bulges, aminoglycoside-based
therapeutics represent potentially potent inhibitors of the miRNA
biogenesis pathway.
Using a fi refl y luciferase reporter system fused to a miRNA-21
target, researchers screened 15 aminoglycosides for their ability to
decrease miR-21, a miRNA overexpressed in multiple cancers [
51
].
Of the molecules, streptomycin acted as a highly effective, selective
inhibitor of miR-21 activity at a concentration comparable to inhi-
bition achieved with antisense LNAs against miR-21 (10
M).
Moreover, the presence of streptomycin also resulted in increased
levels of PDCD4, a known target of miR-21. Interestingly, a closely
related aminoglycoside, dihydrostreptomycin, had no effect on
PDCD4 levels in the cells. Thermal melting experiments were used
μ
