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Fluorescent biosensors can report on the relative abundance, activity, or conformation of biomolecules and analytes through changes in fluorescence emission. A wide variety of genetically-encoded and synthetic biosensors have been developed to monitor protein kinase activity. We have focused on the design, engineering and characterization of fluorescent peptide biosensors of cyclin-dependent kinases (CDKs) that constitute attractive cancer biomarkers and pharmacological targets. In this chapter, we describe the CDKACT fluorescent peptide biosensor technology and its application to assess the relative kinase activity of CDKs in vitro, either using recombinant proteins or cell extracts as a more complex source of kinase. This technology offers a straightforward means of comparing CDK activity in different cell lines and evaluating the specific impact of treatments intended to target kinase activity in a physiologically relevant environment.

The dramatic acceleration in identification of new nucleic acid-based therapeutic molecules such as short interfering RNA (siRNA) and peptide-nucleic acid (PNA) analogues has provided new perspectives for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of nucleic acids together with the low permeability of the cell membrane to negatively charged molecules remain major obstacles to their clinical development. Several non-viral strategies have been proposed to improve the delivery of synthetic short oligonucleotides both in cultured cells and in vivo. Cell-penetrating peptides constitute very promising tools for non-invasive cellular import of oligonucleotides and analogs. We recently described a non-covalent strategy based on short amphiphatic peptides (MPG8/PEP3) that have been successfully applied ex vivo and in vivo for the delivery of therapeutic siRNA and PNA molecules. PEP3 and MPG8 form stable nanoparticles with PNA analogues and siRNA, respectively, and promote their efficient cellular uptake, independently of the endosomal pathway, into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. This chapter describes easy-to-handle protocols for the use of MPG-8 or PEP-3-nanoparticle technologies for PNA and siRNA delivery into adherent and suspension cell lines as well as in vivo into cancer mouse models.

This review focuses on two peptides, MPG and Pep-1, which facilitate the transfer of nucleic acids and proteins, respectively, into subcellular compartments. We have investigated the interactions between these two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration and compression experiments reveal that both peptides interact strongly with phospholipids, and observations on Langmuir-Blodgett transfers indicate that they can modify the lipid organization. Conformational investigations indicate that the lipid-peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG it is based on the formation of a beta-barrel pore-like structure while for Pep-1 it is based on association of helices.