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crc press - E-Lib FK UWKS

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10 Cell-Penetrating Peptides: Processes and Applications<br />

(a)<br />

FIGURE 1.4 Internalization of a Rhodamine-labeled ODN-Tat CPP in HeLa cells. Tat-SS-<br />

5′-ODN-3′-Rhodamine conjugate was incubated with HeLa cells for 30 min at 37°C. Cells<br />

were fixed and the intracellular fluorescence distribution was observed by fluorescence microscopy.<br />

(a) Hoescht staining showing the cell nuclei; (b) immunofluorescence detection of the<br />

chimera, indicating nuclear accumulation.<br />

R.L. Juliano. 28,29 We reasoned that this would be the ideal situation to allow intracellular<br />

release of a minimally modified antisense ODN and thus avoid the risk of<br />

changing hybridization properties.<br />

An alternative strategy would consist in stable conjugation (through a thioether<br />

or an amide bond, for example) of the antisense ODN to the carrier Tat peptide.<br />

Cationic peptide conjugation should indeed accelerate hybridization by increasing<br />

the effective ODN concentration at the target sequence. 31 Whether this will occur at<br />

the expense of diminished sequence-specific recognition should be verified.<br />

A second class of medium-size biomolecules of interest to us consisted of<br />

nonpermeant peptides. Recall that biological processes (for instance, intracellular<br />

signaling cascades) often involve complex networks of protein–protein interactions.<br />

Rapid technological developments in functional genomics have allowed the identification<br />

of peptide domains involved in protein–ligand recognition in increasing<br />

number, thus paving the way for design of peptidic competitors. A severe limitation<br />

to the use of peptides in intact cells and, as a consequence, to their development as<br />

(b)

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