Ribonucleic acids (RNA) are essential biopolymers in biological systems. Initially thought to be an unstable species that acts as a messenger in the flow of genetic information, several key discoveries in the past century have found RNA to contribute to critical functions such as regulation of gene expression and as a catalytic species. Therefore, there is a huge interest in developing tools to further elucidate our understanding of the role of RNA in biological systems, as well as tools to study the structural and functional features of RNA molecules. This thesis presents my efforts in the development of tools for understanding RNA from three perspectives – tracking the expression of RNA in biological systems, probing RNA structure, and discovery of catalytic RNA.The study of response to stimuli by biological systems is a fundamental question in biological sciences. Studies of changes in RNA expression in cellular stimuli response have been lacking due to the limited tools to track RNA expression with temporal resolution. RNA metabolic labeling can label RNA expressed by cells via treatment with an unnatural nucleoside that can be incorporated into cellular RNA. These unnatural nucleosides contain a chemical handle that can be used to chemically modify nascent RNA. However, the use of multiple unnatural nucleosides with selective chemical conjugation strategies has yet to be established in the field. In the first chapter of this thesis, I discuss my work on the discovery of orthogonal chemical reactions to selectively conjugate vinyl-modified nucleosides using a cycloaddition reaction and a phosphine conjugate addition reaction. This method allows the simultaneous use of multiple modified nucleosides with the possibility to selectively tag populations of RNA containing a specific vinyl nucleoside. In the second chapter, I discuss my work on extending the use of the cycloaddition reaction reported in Chapter 1 for sequencing RNA containing 5-vinyluridine (5-VU). Here, a reverse transcriptase (RT) enzyme with Mn2+ buffer was observed to add mutations across the 5-VU site in RNA during RNA-dependent DNA synthesis, therefore allowing the identification of metabolically labeled RNA via mutational profiling. RNA species form complex structures with various intramolecular interactions such as hydrogen bonding and π stacking to stabilize these structures. While there are several biochemical methods to probe the secondary structure of RNA, the tertiary structure of RNA can only be determined by complex analytical methods such as nuclear magnetic resonance or crystallography. Psoralen is an aromatic molecule capable of crosslinking two interacting RNA strands and can be used to interrogate tertiary interactions in RNA structure. In the third chapter, I discuss my efforts in the development of a simplified biochemical method to identify psoralen RNA crosslinks via template switching of RT enzymes. Finally, the fourth chapter presents my efforts in the development of an assay for the discovery of catalytic RNA (ribozyme) from random sequence populations. Although several reports on the selection of various ribozyme species exist, most studies are unable to select RNA that can perform multi-turnover catalysis. This is due to inherent biases in most reports for efficient single turnover as the product is covalently attached to the active sequence and multiple covalent attachments of the product are not possible in most cases. In a novel approach, the assay I developed will allow the selection of multi-turnover catalytic RNA by colocalization of synthesized products and active RNA species, circumventing covalent attachment of the product and ribozyme for the potential discovery of multi-turnover RNA catalysts in a single in vitro selection process.