Biology Reference
In-Depth Information
miRNA precursor (pre-miRNA). Peptide Nucleic Acids were
chosen as oligonucleotide analogs due to their high binding
affi nity and specifi city toward RNA and high stability to degrada-
tion [
7
,
8
]. Molecules able to bind to pre-miRNA affect the pre-
miRNA processing and cause a reduction in the amount of mature
miRNA produced and also pri-miR [
9
]. The case described refers
to inhibition of pre-miR-210 maturation in human leukemia K562
cells, in which over-expression of miR-210 is induced in a time-
dependent and dose-dependent fashion by mithramycin [
10
]. The
strategy of pre-miR targeting is of general application, although it
has to be considered that the protocols for the induction of miR
expression depend on the cell system employed. The general crite-
ria for the design of PNA anti-pre-miR are described. Protocols for
the synthesis of the PNA-based molecules, evaluation of the PNA
cellular uptake by FACS, quantifi cation of pri-miR and mature
miR are described.
The use of pre-miR targeting molecules has potential applica-
tions in the development of miRNA inhibitors in biomedicine and
might also help discovering new details in the structural prefer-
ences and requirements necessary for the assembly of pre-miR pro-
cessing complexes as miRLC and miDPC.
2
Materials
1. PAL-PEG resin LL (Applied Biosystems).
2. Dimethylformamide (DMF).
3. Dichloromethane (DCM).
4. Diethyl ether.
5. Fmoc-protected amino acids.
6. Fmoc-6-Ahx-OH.
7. 4-Methylmorpholine (NMM).
8. Pyridine.
9. Trifl uoroacetic acid (TFA).
2.1 Synthesis
( See Notes 1-3 )
10. m-cresol.
11. Tri-isopropylsilane (TIS).
12. Fluorescein isothiocyanate isomer I (FITC).
13. Fmoc PNA monomers.
14. 0.22 M HOBT/HBTU solution: dissolve 166.9 mg of HBTU
and 67.4 mg of HOBT in 2.0 mL of dry DMF. Vortex the
solution until it is clear.
15. Capping solution: 15 % acetic anhydride, 15 % DIPEA, 70 %
dry DMF (v/v).
16. Deblock solution: 30 % piperidine, 70 % dry DMF (v/v).
