Strategies for Enrichment of Functional RNAs

Ribonucleic acids have complex shapes and versatile functions in biology. This dissertation begins with an introduction on RNA structure and function. RNA also interacts with other molecules such as ions, proteins, and RNAs. Due to the inspiration of evolution of RNA in nature, in vitro selection of RNAs for functions such as binding a target molecule (aptamer) or catalyzing chemical reactions (ribozyme), were also discovered. In chapter 1, I briefly review strategies for probing RNA-RNA interactions, and in vitro selection of RNA aptamers for proteins. Chapter 2 focuses on developing a strategy for mapping RNA-RNA interactions in living cells, particularly the enrichment of trans RNA pairs. I discuss the experiments performed to optimize psoralen crosslinking of RNAs in vitro, followed by the novel approach of dual metabolic labeling of the transcriptome in vivo. The experiments will further aid the development of our strategy for probing the RNA network. Chapter 3 discusses a strategy that was developed to improve selections to yield functional RNAs, by the enrichment of structured monomers from a starting pool. I contributed to this study by doing in vitro selections of RNA aptamers for NanoLuc/Ni-NTA from a starting pool comprised of both the preselected structured pool, and a random pool, which the selection reveals to contain a conserved stem-loop motif that enriched from the structured pool.Chapter 4 will focus on the preliminary experiments to characterize the minimal construct containing the stem-loop motif that was selected to bind Ni-NTA (chapter 3). The experiments include structural probing and column binding assays in the presence of free or chelated nickel and lanthanide ions. We also show that tagging transcripts in vitro with the Ni-NTA motif can be captured on Ni-NTA beads similar to the purification approach of His6-tagged proteins. The chapter concludes with a perspective on future experiments needed for further characterization of the Ni-NTA motif.