Biology Reference
In-Depth Information
2.2 Cell Lines
and Culture
1. K562 cells.
2. Tri-Reagent
TM
.
3. RPMI 1640 medium supplemented with 10 % fetal bovine
serum, 50 U/mL penicillin, and 50 U/mL streptomycin.
4. U6 snRNA.
5. PBS 1×.
6. 1 mM Mithramycin (MTH) stock solution: dissolve 1.085 mg of
mithramycin in 1 mL of ultrapure water/methanol (1:1, v/v).
3
Methods
The PNA oligomer for the pre-miR targeting has to be perfectly com-
plementary to the passenger strand of pre-miRNA, in the region
which is complementary to the mature miRNA. Choose preferentially
the region in which the passenger and the guide strand duplex show
mismatches; this will help the local opening of the RNA duplex and
the displacement of the complementary guide strand. The length of
the PNA oligomer has to be chosen taking in consideration the base
composition of the pre-miR and the stability of the duplex which will
be formed between PNA and RNA. The PNA/RNA duplex has to be
stable at the temperature at which the experiments will be carried out.
Always design also mismatched PNA oligomer, containing one or
two mutations to verify the specifi city of miR maturation inhibition.
Importantly, consider that the PNA oligomer will hardly enter
into cells. Conjugation of PNA to carrier molecules is a requisite
for a functional molecule. Peptides as TAT, the Nuclear Localization
Signal (NLS), or polyArg effi ciently deliver PNA into different
type of cells [
11
,
12
].
An example of PNA sequence designed for pre-miR-210 inhi-
bition is reported in Fig.
1
.
3.1 PNA Design
1. PNA-peptide conjugates are obtained by Fmoc solid phase
synthesis on low loading resins as PAL-PEG-PS resin
(0.18 mmol/g), on a 4
3.2 PNA-Peptide
Synthesis by
Fmoc-Chemistry
mol scale following procedures
reported in the literature [
13
,
14
].
2. Synthesize fi rst the peptide following standard procedures [
14
].
3. At the end of the synthesis remove the last Fmoc treating the
resin with the Deblock solution. To verify whether the synthe-
sis was successful, proceed from step 4 to 10 on a small ali-
quote of resin.
4. Wash the resin with DMF, DCM, and diethyl ether and fi nally
dry the resin under vacuum.
5. Treat the dry resin with TFA/m-cresol/TIS 78/20/2 v/v/v
solution 3 h. Use 1 mL solution for 10 mg of resin.
6. Transfer the TFA solution into an eppendorf tube.
μ
