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42 Cell-Penetrating Peptides: Processes and Applications
Steps
1. Grow cells on glass coverslips; 10 4 to 5 × 10 4 cells/well in a 24-well plate
at the time of coupled oligonucleotide addition is usually appropriate.
2. Dilute the coupled oligonucleotide product in fresh culture medium
(500 µl/well) at a final concentration of 100 nM and add it to the cells
after removal of the culture medium. For coupled peptides, dilute the
product at a final concentration of 10 µM and 30 µgl/ml. DNase I can be
added to the culture medium during peptide incubation in order to reduce
nonspecific binding of the vector to DNA released from dead cells.
3. Incubate for 2 h in normal culture conditions.
4. Wash the cells twice with fresh culture medium.
5. Wash the cells briefly with PBS 500 mM NaCl (to reduce nonspecific
binding on the cell surface).
2.5.1.3 Detection of Internalized AntpHD or Penetratin
2.5.1.3.1 Detection of Peptides in Live Cells
This kind of observation is possible only with fluorochrome-labeled peptides; in that
case, live cells are directly observed under a fluorescence microscope and can
alternatively be sorted isolated with a FACS-Scan.
2.5.1.3.2 Detection of Biotin-Labeled Peptides
1. Fix cells in a solution of ethanol/acetic acid (9:1) for 5 min at –20°C.
2. Wash the cells three times for 5 min with PBS.
3. Incubate the cells in PBS 10% NCS for 30 min at room temperature.
4. Wash the cells three times for 5 min with PBS.
5. Incubate the cells with FITC-streptavidin in PBS 10% NCS for 30 min
at room temperature.
6. Wash the cells three times for 2 min with PBS.
7. Wash the cells with distilled water.
8. Air-dry the coverslips and mount them in antifading medium (DAKO)
before examination with a fluorescence microscope.
2.5.1.3.3 Detection of Radioactive Peptides Detection
Count directly with a scintillation counter.
2.5.2 ANTPHD AND PENETRATIN-COUPLED CARGOES
2.5.2.1 Cargo–Vector Linkage
2.5.2.1.1 Oligonucleotides and PNAs
The principle of the coupling reaction is the formation of a disulfide bond between
the vector and the cargo molecules (Figure 2.2). At one extremity the vector contains
a disulfide pyridinium function that is highly reactive when incubated with free SH
groups but which cannot react with itself, thus preventing the formation of
homodimers. Penetratin-l is supplied in this form (activated penetratin-1), otherwise
the function can be added at the N-terminal end of the peptide during synthesis (e.g.,