Structural Relay Methods for Functional Peptide Discovery

描述(申请人提供)：寡肽在生物学中有许多功能，包括提供结合选择性，允许蛋白质在细胞内、细胞间、组织内和整个生物体中的适当位置进行运输。这种转运功能对新的治疗和诊断试剂的开发变得越来越重要，因为对非靶标效应的控制要求变得越来越严格，输送的试剂也变得越来越昂贵。然而，人口贩运很难做到，主要是因为它是一个多元问题，因此超出了我们理性设计的能力。我们建议将强大的分子进化工具应用于不携带遗传信息的代理人的交付。我们将开发一种新的三步法来识别携带所需货物进入细胞或特定细胞细胞器的多肽。该方法依赖于SELEX技术(通过指数富集法对配体的系统进化)创建选择性与任何特定多肽结合的DNA序列的能力，以及噬菌体展示识别与任何DNA分子结合的多肽的能力。有了这样的工具，我们将准备与感兴趣的货物结合的多肽文库，并将它们与感兴趣的细胞孵育。可以预期，该分子群体中的一小部分会流向所需的目的地，例如细胞质、线粒体或细胞核。这种细胞物质的分离完成了所需多肽(S)与其余候选多肽的分离，但数量非常少。目前还没有一种技术可以在货物没有对多肽的身份进行编码的情况下识别这些多肽。我们希望SELEX和噬菌体展示技术的连续使用将允许检测到功能性多肽，并将它们的身份信息“放大”，以便能够对它们进行鉴定、重新合成和测试。如果成功，这些研究将导致在广泛的环境和应用中发现功能寡肽的通用方法。

DESCRIPTION (provided by applicant): Oligopeptides have many functions in biology, including providing binding selectivity that allows proteins to traffic to the proper place in the ell, between cells, within tissues, and throughout the organism. This trafficking function is becoming more and more important to the development of new therapeutic and diagnostic agents, as the requirements for the control of off-target effects are becoming ever more stringent and the delivered agents ever more expensive. However, trafficking is hard to do, mostly because it is a multivariate problem, and so is beyond our capabilities of rational design. We propose to bring powerful tools of molecular evolution to bear on the delivery of agents that do not carry genetic information. We will develop a new three-step method to identify peptides that carry desired cargoes into cells or to particular cellular organelles. The method relies on the ability of the SELEX technique (systematic evolution of ligands by exponential enrichment) to create DNA sequences that bind selectively to any particular peptide, and the ability of phage display to identify peptides that bind to any DNA molecule. With such tools in hand, we will prepare libraries of peptides bound to the cargo of interest, and incubate them with cells of interest. A very small fraction of that molecular population can be expected to traffic to the desired destination, such as the cytoplasm, mitochondria, or nucleus. Isolation of that cellular material accomplishes the separation of the desired peptide(s) from the rest of the candidates, but in very small amounts. No technique is currently available that will allow one to identify such peptides if the cargo does not code for the identity of the peptide. We hope that the sequential use of the SELEX and phage display techniques will allow the functional peptides to be detected and the information of their identities "amplified" so that they can be identified, resynthesized, and tested. If successful, these studies should lead to a general method to discover functional oligopeptides in a wide variety of settings and applications.