Enhanced Delivery of Protein Biosensors: a Combinatorial Library Strategy

描述(申请人提供)：与荧光染料结合的蛋白质生物传感器在成像关键的细胞内过程中发挥着至关重要的作用。然而，目前将这些生物传感器输送到适当的亚细胞室的方法是粗糙和无效的。在这里，我们提出了一种新颖而有力的方法来解决这个问题。我们将使用腺相关病毒载体中表达的多肽组合文库来筛选能够促进蛋白质生物传感器在细胞内高效传递的多肽(即所谓的细胞穿透性多肽，CPPs)。我们将对进入细胞并在胞浆中积累的多肽特别感兴趣，因为许多生物传感器都是为了在胞浆中发挥作用而设计的。从病毒筛选中出现的候选多肽(CPP)将进一步测试其固有的进入细胞的能力以及有效传递基于染料的蛋白质生物传感器的能力。前景看好的CPP将被用于与蛋白质生物传感器形成嵌合体。将使用各种技术来评估CPP/生物传感器嵌合体的总细胞积累和定量亚细胞分布。我们预计，这种方法将导致一批新的CPP，这将对整个蛋白质细胞内传递领域产生重要影响，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.