Protein Labeling and Crosslinking by Covalent Aptamers

项目总结 这项提议的总体目标是开发一种全新的寡核苷酸制剂--共价 适配子--这将对基于核酸的试剂和疗法产生重大影响。共价适配子 将成为新适体应用程序背后的启用方法，并将提供新的持久解决方案 此复合类的限制。适配子是一种经过改造的寡核苷酸，可以结合蛋白质靶标并具有 在许多领域都有应用，包括治疗学、诊断学、药物输送和成像。他们 以与抗体相媲美的亲和力和特异性结合靶标，同时表现出更低的产量 成本，更高的生产一致性，以及轻松引入化学修饰的能力。利用 扩增和测序核酸的能力，适体是通过SELEX产生的，SELEX是一种强大的 体外选择过程。我们的中心假设是适体之间形成的共价键-或 由适配子传递的功能基序及其目标蛋白将提供前所未有的停留时间， 增加核酸酶的稳定性(或不需要核酸酶)，并促进适体的选择。此外，共价 键的形成使基于适体平台的新应用成为可能。以下目标将考验这一点- 假肢。目的1：共价适配子的化学。对设计有全面的了解 共价适配子背后的原理并使其具有广泛的适用性，我们将基于以下内容探索它们的化学 我们有希望的初步结果。我们将探索在抗体药物偶联组装中的应用，以及 催化共价键的形成。目的2：基于细胞的共价适配子的应用。我们将开发几个 由共价适配子实现并展示其作为化学生物探针的用途的应用。 由于已经报道了丰富的适体集合，我们将重点放在细胞表面蛋白上。目标3： 共价适配子的选择。对于能够选择性地交叉结合的共价适配子的从头产生 在没有先前序列和/或结构信息的情况下，连接到任何选定的蛋白质靶标，我们将开发一种基因-- 实现了体外选择过程，为传统的SELEX增加了明显的优势。

PROJECT SUMMARY The overall goal of this proposal is to develop a fundamentally new class of oligonucleotide agents – covalent aptamers – that will have a major impact on nucleic acid-based reagents and therapeutics. Covalent aptamers will be an enabling methodology behind new aptamer applications and will provide new solutions to persistent limitations of this compound class. Aptamers are engineered oligonucleotides that bind protein targets and have found applications in a multitude of areas, including therapeutics, diagnostics, drug delivery, and imaging. They bind their targets with affinity and specificity that rivals those of antibodies, while displaying lower production cost, higher production consistency, and the ability to easily introduce chemical modifications. Capitalizing on the ability to amplify and sequence nucleic acids, aptamers are being generated through SELEX, a powerful in vitro selection process. Our central hypothesis is that the formation of a covalent bond between an aptamer – or a functional motif delivered by the aptamer – and its target protein will provide unprecedented residence time, increase nuclease stability (or obviate the need for it), and facilitate aptamer selection. Furthermore, covalent bond formation enables new applications based on the aptamer platform. The following aims will test this hy- pothesis. Aim 1: The chemistry of covalent aptamers. To gain a comprehensive understanding of the design principles behind covalent aptamers and to enable broad applicability, we will explore their chemistry based on our promising preliminary results. We will explore applications in antibody drug conjugate assembly, as well as catalytic covalent bond formation. Aim 2: Cell-based applications of covalent aptamers. We will develop several applications that are enabled by covalent aptamers and that showcase their utility as chemical biology probes. We are focusing on cell-surface proteins due to the rich collection of aptamers that have been reported. Aim 3: Selection of covalent aptamers. For the de novo generation of covalent aptamers capable of selectively cross- linking to any chosen protein target without prior sequence and/or structural information, we will develop a gen- eralized in vitro selection process that adds distinct advantages to traditional SELEX.