Protein Labeling and Crosslinking by Covalent Aptamers

共价适体的蛋白质标记和交联

• 批准号: 10391640
• 负责人: Alexander Deiters
• 金额: $ 35.19万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-02 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10391640
• 关键词: Address; Affinity; Amino Acids; Animal Model; Antibodies; Antibody-drug conjugates; Antisense Oligonucleotides; Area; Binding; Binding Proteins; Biological; Biological Models; Biology; Cell Surface Proteins; Cell model; Cell surface; Cells; Chemicals; Chemistry; Collection; Data; Detection; Development; Diagnostic; Disease; Drug Delivery Systems; Embryo; Engineering; Evaluation; Generations; Goals; Government; Image; Immobilization; Immune Targeting; Immune response; In Vitro; Kinetics; Label; Libraries; Ligands; Literature; Mediating; Methodology; Modification; Mus; Nucleic Acids; Nucleotides; Oligonucleotides; Persons; Positioning Attribute; Post-Translational Protein Processing; Preparation; Process; Production; Proteins; RNA vaccine; Reaction; Reagent; Reporting; Research; Research Personnel; SARS-CoV-2 spike protein; Site; Specificity; Structure-Activity Relationship; Testing; Therapeutic; Thrombin; Time; Virus; Zebrafish; aptamer; base; circulating cancer cell; cost; covalent bond; crosslink; design; experience; improved; in vivo; novel; nuclease; nucleic acid-based therapeutics; programs; protein degradation; residence; small molecule inhibitor; success; tool; transcription factor

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.

项目摘要 该提案的总体目标是开发一类新的寡核苷酸代理 - 共价 适体 - 这将对基于核酸的试剂和治疗产生重大影响。共价适体 将是新的Apater应用程序背后的一种有利方法，并将提供新的解决方案以持续 该化合物类的局限性。 APATMER是结合蛋白质靶标并具有的工程寡核苷酸 在许多领域发现了应用，包括治疗，诊断，药物输送和成像。他们 以相关性和特异性绑定抗体的目标，同时表现出较低的产量 成本，更高的生产一致性以及轻松引入化学修饰的能力。大写 通过SELEX产生了放大和顺序的核酸，适体的能力，这是一种强大的 体外选择过程。我们的核心假设是，apatmer或 置换者提供的功能基序及其靶蛋白将提供前所未有的停留时间， 提高核酸酶稳定性（或消除对其的需求），并促进纠正措施选择。此外，共价 债券形成可以基于Apater平台实现新应用程序。以下目的将测试此问题 pothesis。目标1：共价适体的化学。要全面了解设计 共价适体背后的原则并实现广泛的适用性，我们将基于 我们承诺的初步结果。我们将探索抗体药物共轭组装中的应用，以及 催化共价形成。 AIM 2：共价适体的基于细胞的应用。我们将开发几个 通过共价适体启用的应用，并展示其作为化学生物学问题的实用性。 由于已经报道的丰富的适体收集，我们将重点放在细胞表面蛋白上。目标3： 选择共价适体。对于从头产生的共价适体，能够选择性交叉 在没有事先序列和/或结构信息的情况下链接到任何选择的蛋白质靶标，我们将开发一个gen- 在体外选择过程中，为传统的SELEX增添了不同的优势。