Structural Relay Methods for Functional Peptide Discovery

描述（由申请人提供）：寡肽在生物学中具有许多功能，包括提供结合选择性，允许蛋白质运输到细胞、细胞之间、组织内和整个生物体中的适当位置。随着对控制脱靶效应的要求越来越严格和所递送的药物越来越昂贵，这种贩运功能对开发新的治疗和诊断药物变得越来越重要。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。我们建议将分子进化的强大工具用于不携带遗传信息的药物的递送。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器的肽。该方法依靠SELEX技术（通过指数富集的配体系统进化）的能力来创建选择性结合任何特定肽的DNA序列，以及噬菌体展示识别结合任何DNA分子的肽的能力。有了这些工具在手，我们将准备与感兴趣的货物结合的肽库，并与感兴趣的细胞孵育它们。这些分子群中的很小一部分可以被运送到预期的目的地，如细胞质、线粒体或细胞核。该细胞物质的分离实现了所需肽与其他候选肽的分离，但数量非常少。如果货物不编码肽的身份，目前没有可用的技术允许人们识别这种肽。我们希望连续使用SELEX和噬菌体展示技术将允许功能肽被检测，其身份信息被“放大”，以便它们可以被识别，重新合成和测试。如果成功，这些研究将导致在各种设置和应用中发现功能性寡肽的通用方法。

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. PUBLIC HEALTH RELEVANCE: Modern drug development requires molecules that can deliver therapeutic agents to specific cells in the body. We propose to test a new method for discovering peptide molecules that have this ability, using a combination of chemical synthesis and methods of molecular evolution. The resulting technique should allow almost any drug to be targeted to any desired biological destination.