Protein biosensors conjugated with fluorescent dyes play a vital role in imaging key intracellular processes. However current methodologies for the delivery of these biosensors to appropriate subcellular compartments are crude and ineffective. Here we propose a novel and powerful approach to solving this problem. We will use peptide combinatorial libraries expressed in an Adeno Associated Virus vector to screen for peptides (so called 'cell penetrating peptides', CPPs) able to promote efficient intracellular delivery of protein biosensors. We will be particularly interested in peptides that enter cells and accumulate in the cytosol rather than in endomembrane compartments, since many biosensors are designed to function in the cytosol. Candidate peptides (CPPs) emerging from the viral screen will be further tested for innate ability to enter cells and for the ability to effectively deliver dye-based protein biosensors. Promising CPPs will be used to form chimeras with protein biosensors. A variety of techniques will be used to evaluate total cellular accumulation and quantitative subcellular distribution of the CPP/biosensor chimeras. We anticipate that this approach will lead to a cohort of novel CPPs that will have an important impact on the entire field of intracellular delivery of proteins, especially biosensors. ? ? ?
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