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种全新的寡核苷酸试剂-共价寡核苷酸。 适体-这将对基于核酸的试剂和疗法产生重大影响。共价适体 将是新适体应用背后的一种使能方法，并将为持久性 这个复合类的局限性。适体是工程化的寡核苷酸，其结合蛋白质靶标并具有 在包括治疗学、诊断学、药物递送和成像在内的众多领域中找到了应用。他们 与靶点结合的亲和力和特异性与抗体相当， 成本、更高的生产一致性以及容易引入化学改性的能力。充分利用 核酸扩增和测序的能力，适体是通过SELEX产生的，一种强大的插入技术， 体外选择过程。我们的中心假设是，适体-或 由适体及其靶蛋白递送的功能基序将提供前所未有的停留时间， 增加核酸酶稳定性（或减少对它的需要），并促进适体选择。此外，共价 键的形成使得基于适体平台的新应用成为可能。以下目标将检验这一假设- 假设目的1：共价适体的化学。全面了解设计 共价适体背后的原理，并使广泛的适用性，我们将探索他们的化学基础上， 我们有希望的初步结果。我们将探索抗体药物偶联物组装中的应用，以及 催化共价键形成。目的2：共价适体的基于细胞的应用。我们将开发几个 这些应用是由共价适体实现的，并展示了它们作为化学生物学探针的实用性。 我们专注于细胞表面蛋白质，因为已经报道了丰富的适体集合。目标3： 共价适体的选择。对于能够选择性交叉的共价适体的从头生成， 连接到任何选定的蛋白质靶点，而无需事先的序列和/或结构信息，我们将开发一个基因， 一种成熟的体外选择过程，为传统的SELEX增加了明显的优势。