Inhibition of miRNA Maturation by Peptide Nucleic Acids - miRNA Maturation: Methods and Protocols - page 154

Fig. 1 Representation of the pre-miRNA-210 and the designed PNA anti-pre-miR (reproduced from ref. [ 9 ] )

7. Evaporate TFA by nitrogen insuffl ation.
8. Add cold diethyl ether to precipitate the oligomer; keep the
eppendorf in ice for 5 min; centrifuge 3 min at 11,000 rpm
(8,000 × g ). Pour the ether in a separate tube and wash the
precipitate again with ether.
9. Dissolve the crude in the minimum volume of H 2 O and
lyophilize it.
10. Analyze the crude by LC-MS.
11. Synthesize the PNA oligomer on the resin anchored peptide
following standard procedures [ 13 ]. At the end of the synthe-
sis cleave the conjugate off the resin, lyophilize the crude and
HPLC purify the product ( steps 3-10 ). Check the identity of
the molecule by mass spectroscopy and its purity by HPLC.

3.3 Synthesis of the
FITC-PNA- Peptide
Conjugates

For the synthesis of the FITC derivative, use the PNA-peptide
conjugates anchored to the resin, with the free N-terminal amino
group ( see Note 4 ).
1. Swell the resin in DMF for 1 h.
2. Dissolve 7.06 mg of Fmoc-

ε -Ahx-OH in a 89.1 μ L of 0.22 M

L of NMM.
Add the solution to the resin and let the reaction proceed for
40 min.
3. Wash the resin with DMF.
4. Add 0.5 mL of capping solution and let the reaction proceed
for 5 min.
5. Wash extensively the resin with DMF.
6. Treat the resin with the deblock solution for 5 min (2 times).
7. Protect the reaction vessel from the light ( see Note 3 ).
8. In a dark tube dissolve 15.56 mg of FITC in 200

solution of HOBT/HBTU in DMF and add 3.08 μ

μ L of DMF.

Add 6.17 μ L of NMM. Transfer the solution to the reaction