Biology Reference
In-Depth Information
the footprinting experiment, inhibiting Dicer processing of the
pre-miRNA. However, actual testing of maturation was not exam-
ined, nor was the specifi city of the HTP towards other similar pre-
miRNAs. The generation of a larger library of macrocyclic HTPs
to increase specifi city in conjunction with the in vivo assessment of
these compounds may lead to viable inhibitors of specifi c pre-
miRNA maturation.
In an alternative approach by Yu and co-workers, amphiphilic
helical peptides containing acridines were employed as potential
pre-miRNA binders [
48
]. In order to enhance binding affi nity and
specifi city to different target RNA motifs conjugated ligands con-
taining a variety of binding modes were utilized. An acridine moi-
ety was selected to intercalate, while the peptide targets the RNA
stem regions. An array of ten amphiphilic peptides with differing
α
-helical content and an acridinylated residue at variable positions
was measured for a binding to different RNA substrates (including
pre-miR-24-1) using fl uorescence anisotropy. The position of the
acridine moiety was found to signifi cantly affect the conforma-
tional rigidity, and resulted in differential binding of RNA targets
at nanomolar concentrations. By increasing the number of acridi-
nylated residues while maintaining the helicity, the specifi city for
different targets was enhanced to ~250 pM. However, the specifi c-
ity for different analogs of pre-miR-24-1 was not discernable, and
no other miRNA targets were examined. While this approach is
promising, it is still unclear whether the necessary specifi city
towards a variety of pre-miRNAs can be achieved, and the ability of
helical amphiphilic peptides to actually modulate miRNA matura-
tion has yet to be demonstrated either in vivo or in vitro.
Finally, the Luebke laboratory demonstrated the in vitro speci-
fi city of a peptoid library to bind pre-miR-21 using a fl uorescence
assay [
49
]. Peptoids are useful targeting agents due to their cell
permeability, protease resistance, chemical diversity, and ease of
synthesis [
50
]. Additionally, the ability to screen these molecules in
a microarray format affords the ability to examine thousands of
compounds simultaneously as replicate arrays can be printed from
a single chemical library. This feature allows for the screening of
the same library against multiple targets. A spatially isolated pep-
toid library was constructed on a solid support from a chemically
diverse set of 21 monomers theoretically representing 9,261 com-
pounds, all with a C-terminal arginine to enhance RNA binding.
The peptoid microarrays were then incubated in the presence of
fl uorescently labeled pre-miR-21 or a fl uorescently labeled control
RNA to ascertain differential versus nonspecifi c binding. Positive
binding compounds were identifi ed by fl uorescence microscopy
based on reproducibly detectable fl uorescence in the pre-miR-21
conditions versus a control RNA hairpin (Fig.
5
). Two peptoids
demonstrated specifi c binding activity with dissociation constants
of approximately 2
μ
M and 20-fold discrimination between the
