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

促进单个RNA研究的工具对于理解它们的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、丰度和寿命都不同。该项目旨在设计新的酶，将新的小分子记者插入到RNA中，以适合研究任何感兴趣的RNA的方式。这些酶将插入小到不会干扰RNA功能的报告，并以特定于单个RNA的方式这样做。这些前所未见的酶和小分子将被编程为与天然酶和小分子一起工作，而不会出现不必要的串扰，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它们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上的介绍提高有关遗传机制的社会素养，以及通过当地高中的外联活动增加对代表性不足群体的STEM接触。这项研究计划利用定向进化来发现新的RNA修饰酶，该酶能够将RNA中的核苷酸碱基交换为功能上的小分子报告分子。定向进化工作流程将包括反复几轮的位点饱和突变和一个独特的高通量筛选，以探测突变的转糖基酶接受新底物的能力。这项技术避免了最先进技术的限制，包括庞大的记者、瞬时关联和脱离目标安装。这种酶将进化成与内源RNA修饰酶及其底物垂直作用，从而促进高保真标记和低本底。这种RNA修改策略的多功能性将为调查人员提供一个前所未有的工具来研究他们的选择成绩单。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.