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。该项目旨在设计新的酶，以适合研究任何感兴趣的 RNA 的方式将新型小分子报告基因插入 RNA 中。这些酶将插入足够小的报告基因，不会干扰 RNA 功能，并以特定于单个 RNA 的方式插入报告基因。这些前所未见的酶和小分子将被编程为与它们的天然对应物一起工作，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进行展示来提高有关遗传机制的社会素养，并通过当地高中的推广活动来增加代表性不足群体的 STEM 曝光率。该研究项目打算利用定向进化来发现新的 RNA 修饰酶，能够将 RNA 中的核碱基交换为功能性小分子报告基因。定向进化工作流程将涉及迭代的位点饱和诱变和独特的高通量筛选，以探测突变型转糖基酶接受新底物的能力。该技术避免了最先进技术的限制，包括笨重的报告器、瞬时关联和脱靶安装。该酶将进化为与内源性 RNA 修饰酶及其底物正交地起作用，从而促进高保真度标记和低背景。 这种 RNA 修饰策略的多功能性将为研究人员提供前所未有的工具来研究他们选择的转录本。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值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.