2.2 Peptide Nucleic
Acids (PNAs)
PNAs can be purchased from several companies, including Panagene Inc. (www.panagene.com). Alternatively, PNAs against miRNAs can be synthesized following the procedures described in
Manicardi et al. [28]. The data here presented are based on PNAs
described in the chapter by Brognara et al. [46]. The sequences of
the different PNAs-a221 used are reported in Table 1. The criteria
for the design of the PNAs for increasing selectivity of hybridization
and low nonspecific effects are described in Note 1.
Figure 1 shows possible targeting of anti-apoptotic PUMA
3’UTR mRNA region by the employed miR-221-3p (a,b). Full
complementarity does exist between miR-221-3p and the antimiR PNA-a221 (sequences reported in Table 1). Some of the
best characterized biological targets are also shown (c) [65–72].
2.3 Cell Culture
1. Human glioma U251 cell line [64] (Sigma-Aldrich, St.Louis,
Missouri, USA).
2. RPMI 1640 medium.
3. 100 U/mL penicillin and 100 μg/mL streptomycin, final
concentration.
4. Fetal bovine serum.
2.4 Cellular Uptake
1. FACScan (BD, Becton Dickinson, Franklin Lakes, New
Jersey, USA).
2. 1Â Dulbecco’s PBS (DPBS).
3. CellQuest Pro Software (BD, Becton Dickinson, Franklin
Lakes, New Jersey, USA).
2.5 RNA Extraction
1. Trypsin-EDTA.
2. FBS.
3. DPBS.
4. Tri-Reagent to lyse cells.
5. Nuclease-free water to resuspend RNA.
Table 1
Sequences of the PNAs employed in the described studies
PNAs
PNA sequence
Target miRNAs
Fl-PNA-a221
Fl-AEEA-AAACCCAGCAGACAATGT-NH 2
miR-221-3p
Fl-R8-PNA-a221
Fl-AEEA-R 8 -AAACCCAGCAGACAATGT-NH 2
miR-221-3p
PNA-a221
H-AAACCCAGCAGACAATGT-NH 2
miR-221-3p
R8-PNA-a221
H-R 8 -AAACCCAGCAGACAATGT-NH 2
miR-221-3p
R8-PNA-a221-MUT
(mutations underlined)
H-R 8 -AATCCCACCAGAGAAAGT-NH 2
miR-221-3p
PNA Delivery with Argininocalix[4]arene
127
Acids (PNAs)
PNAs can be purchased from several companies, including Panagene Inc. (www.panagene.com). Alternatively, PNAs against miRNAs can be synthesized following the procedures described in
Manicardi et al. [28]. The data here presented are based on PNAs
described in the chapter by Brognara et al. [46]. The sequences of
the different PNAs-a221 used are reported in Table 1. The criteria
for the design of the PNAs for increasing selectivity of hybridization
and low nonspecific effects are described in Note 1.
Figure 1 shows possible targeting of anti-apoptotic PUMA
3’UTR mRNA region by the employed miR-221-3p (a,b). Full
complementarity does exist between miR-221-3p and the antimiR PNA-a221 (sequences reported in Table 1). Some of the
best characterized biological targets are also shown (c) [65–72].
2.3 Cell Culture
1. Human glioma U251 cell line [64] (Sigma-Aldrich, St.Louis,
Missouri, USA).
2. RPMI 1640 medium.
3. 100 U/mL penicillin and 100 μg/mL streptomycin, final
concentration.
4. Fetal bovine serum.
2.4 Cellular Uptake
1. FACScan (BD, Becton Dickinson, Franklin Lakes, New
Jersey, USA).
2. 1Â Dulbecco’s PBS (DPBS).
3. CellQuest Pro Software (BD, Becton Dickinson, Franklin
Lakes, New Jersey, USA).
2.5 RNA Extraction
1. Trypsin-EDTA.
2. FBS.
3. DPBS.
4. Tri-Reagent to lyse cells.
5. Nuclease-free water to resuspend RNA.
Table 1
Sequences of the PNAs employed in the described studies
PNAs
PNA sequence
Target miRNAs
Fl-PNA-a221
Fl-AEEA-AAACCCAGCAGACAATGT-NH 2
miR-221-3p
Fl-R8-PNA-a221
Fl-AEEA-R 8 -AAACCCAGCAGACAATGT-NH 2
miR-221-3p
PNA-a221
H-AAACCCAGCAGACAATGT-NH 2
miR-221-3p
R8-PNA-a221
H-R 8 -AAACCCAGCAGACAATGT-NH 2
miR-221-3p
R8-PNA-a221-MUT
(mutations underlined)
H-R 8 -AATCCCACCAGAGAAAGT-NH 2
miR-221-3p
PNA Delivery with Argininocalix[4]arene
127