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.
