Enhanced Delivery of Protein Biosensors: a Combinatorial Library Strategy

描述（由申请人提供）：与荧光染料结合的蛋白质生物传感器在关键细胞内过程成像中发挥重要作用。然而，目前用于将这些生物传感器递送到适当的亚细胞区室的方法是粗糙和无效的。在这里，我们提出了一种新颖而强大的方法来解决这个问题。我们将使用在腺相关病毒载体中表达的肽组合文库来筛选能够促进蛋白质生物传感器的有效细胞内递送的肽（所谓的“细胞穿透肽”，CPP）。我们将特别感兴趣的肽，进入细胞，并积累在胞质溶胶，而不是在内膜室，因为许多生物传感器的功能，在胞质溶胶。从病毒筛选中出现的候选肽（CPP）将进一步测试进入细胞的先天能力和有效递送基于染料的蛋白质生物传感器的能力。有前途的CPP将用于与蛋白质生物传感器形成嵌合体。将使用多种技术评价CPP/生物传感器嵌合体的总细胞蓄积和定量亚细胞分布。我们预计，这种方法将导致一组新的CPP，将有一个重要的影响，对整个领域的细胞内交付的蛋白质，特别是生物传感器。

DESCRIPTION (provided by applicant): 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.