EAGER: Sensors for in vivo Localization and Quantification of Eukaryotic Small RNAs

智力优势：绿色荧光蛋白（GFP）和其他基因编码荧光标签的出现完全改变了科学家监测活细胞内蛋白质的方式。直到最近，RNA分子的研究还缺乏类似的强大工具来监测特定RNA转录物的存在或丰度。然而，结合GFP样发色团的RNA“适体”（具有结合特性的短寡核苷酸）的发展取得了重大进展，将这些检测技术扩展到mRNA甚至代谢物。小RNA（如microRNA和短干扰RNA）的可视化面临着独特的挑战，因为附加的荧光标签要么在加工过程中被去除，要么破坏RNA功能。因此，目前的方法要么间接监测细胞中的小RNA，要么需要破坏性的固定细胞制备，这妨碍了体内研究。该项目将开发一种克服这些限制的小RNA传感器。与基于蛋白质的GFP标记一样，该项目中开发的传感器将被遗传编码，并将使用成熟的荧光显微镜技术可见。传感器仅在小RNA结合时才会发出荧光，并能够监测小RNA的相对和绝对水平。该项目还将优化基于信息学的方法来设计这些检测分子。因此，这项工作的智力价值是开发新的方法，在体内检测小RNA.Broader影响：这个工具的开发有可能改变小RNA的研究方式。小RNA是大多数真核生物中重要的调控分子，并且植物具有特别丰富和复杂的一组用于这些分子的生物合成的途径。真核小RNA的功能非常多样，包括在发育、应激反应和表观遗传调控中的作用，因此它们是分子分析的极其重要的靶标。虽然该传感器将首先被开发和部署用于植物研究，但它将成为研究大多数真核生物（包括动物系统）中小RNA的有力工具。 因此，这项工作旨在提供数据和工具，以深入了解以前无法实现的小RNA的定位，丰度，运动甚至功能。从教育的角度来看，该项目将有助于博士后研究员的专业发展，他们将获得实验，计算和基因组学方法的经验，这些方法是研究的一个组成部分。

Intellectual Merit: The advent of green fluorescent protein (GFP) and other genetically-encoded fluorescent tags has completely changed the way scientists monitor proteins within living cells. Until recently, studies of RNA molecules have lacked a similarly powerful set of tools to monitor the presence or abundance of specific RNA transcripts. However, major advances in the development of RNA "aptamers" (short oligonucleotides with binding properties) that bind GFP-like chromophores have extended these detection techniques to mRNA and even metabolites. The visualization of small RNAs, such as microRNAs and short-interfering RNAs, presents unique challenges because appended fluorescent tags either get removed during processing or disrupt the RNA function. Consequently, current methods either indirectly monitor small RNAs in cells or require disruptive fixed cell preparations, which prevent in vivo studies. This project will develop a small RNA sensor that overcomes these limitations. As with the protein-based GFP marker, the sensors developed in this project will be genetically encoded and will be visible using well-established fluorescent microscopy techniques. The sensor will fluoresce only when a small RNA is bound and will have the ability to monitor relative and absolute levels of small RNAs. The project will also optimize informatics-based approaches for the design of these detection molecules. Thus the intellectual merit of the work is the development of novel methods for the in vivo detection of small RNAs.Broader Impacts: Development of this tool has the potential to transform the way small RNAs are investigated. Small RNAs are important regulatory molecules in most eukaryotes, and plants have a particularly abundant and complex set of pathways for the biogenesis of these molecules. The functions of eukaryotic small RNAs are quite diverse, including roles in development, stress responses, and epigenetic regulation, and thus they are extremely important targets for molecular analysis. Although the sensor will first be developed and deployed for studies in plants, it will be a powerful tool for studying small RNAs in most eukaryotes, including animal systems. Therefore, this work intends to provide both data and tools for insights into the localization, abundance, movement, and even the function of small RNAs that were previously unattainable. From an educational perspective, this project will contribute to the professional development of a postdoctoral fellow, who will gain experience in experimental, computational and genomics methods that are an integral part of the research.