EAGER: Developing a Highly Selective, Orthogonal, Enzymatic RNA Labeling Technology via Directed Evolution of an RNA Transglycosylase

EAGER：通过 RNA 转糖基酶的定向进化开发高度选择性、正交、酶促 RNA 标记技术

• 批准号: 2136169
• 负责人: Neal Devaraj
• 金额: $ 30万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2136169&HistoricalAwards=false
• 关键词: EAGER; Developing; Highly; Selective; Orthogonal

Tools that facilitate the study of individual RNAs are critical for understanding their mechanisms of action and biological significance. RNAs are difficult to study as they vary in size, function, location, abundance, and lifetime. This project intends to engineer new enzymes that insert novel, small molecule reporters into an RNA in a manner that is appropriate for studying any RNA of interest. These enzymes will insert reporters that are small enough to not interfere with RNA function and do so in a manner that is specific to that individual RNA. These never-before-seen enzymes and small molecules will be programmed to work alongside their native counterparts without unwanted crosstalk, allowing researchers to efficiently investigate how location and abundance of RNAs influences their role in genetic mechanisms. The project will have broad impacts including improving societal literacy surrounding genetic mechanisms through presentation in accessible media platforms and increasing STEM exposure of underrepresented groups through local high school outreach. This research project intends to use directed evolution to discover new RNA modifying enzymes capable of exchanging a nucleobase in an RNA for a functional small molecule reporter. The directed evolution workflow will involve iterative rounds of site saturation mutagenesis and a unique high throughput screen to probe mutant transglycosylases for their ability to accept novel substrates. This technology avoids the limitations of state-of-the-art technologies, including bulky reporters, transient association, and off target installation. The enzyme will be evolved to act orthogonally to endogenous RNA modification enzymes and their substrates, facilitating high fidelity tagging and low background. The versatility of this RNA modification strategy will provide investigators with an unprecedented tool to study their transcript of choice.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

促进研究单个RNA的工具对于理解其作用机制和生物学意义至关重要。 RNA很难研究，因为它们的大小，功能，位置，丰度和寿命各不相同。该项目旨在以适合研究任何感兴趣的RNA的方式插入新颖的小分子记者的新酶。这些酶将插入足够小以不干扰RNA功能的记者，并以特定于该单个RNA的方式进行。这些从未见过的酶和小分子将被编程为无需不需要的串扰的天然对应物与其同行一起工作，从而使研究人员能够有效地研究RNA的位置和丰度如何影响其在遗传机制中的作用。该项目将产生广泛的影响，包括通过在无障碍媒体平台中介绍围绕遗传机制的社会素养，并通过当地的高中宣传来增加代表性不足的群体的茎。该研究项目打算使用定向进化来发现能够在RNA中换成核碱基的新RNA，以换成功能性小分子报告基因。定向的进化工作流将涉及位点饱和诱变的迭代回合，以及独特的高吞吐量筛选，以探测突变体透糖基酶，以便他们接受新型底物。该技术避免了最新技术的局限性，包括笨重的记者，瞬态关联和关闭目标安装。该酶将进化为与内源性RNA修饰酶及其底物的正交作用，从而促进高保真标记和低背景。 这种RNA修改策略的多功能性将为研究人员提供一种前所未有的工具来研究其选择的成绩单。该奖项反映了NSF的法定任务，并使用基金会的知识分子优点和更广泛的影响审查标准，认为值得通过评估值得支持。