Book excerpt: ADARs (adenosine deaminases acting on RNA) are enzymes that catalyze the post-transcriptional deamination of adenosine to inosine in double-stranded RNA, a type of RNA editing. Inosine is recognized by the translation machinery as guanosine, so RNA editing can result in incorporation of different amino acids than those encoded in the genome. While some structural information is available for one enzyme in this family, ADAR2, there is a distinct lack of structural information regarding ADAR1. In addition, many questions exist regarding the biological function of these enzymes. In recent years new substrates for these enzymes have been identified, but their role is unknown. This dissertation describes experiments in which we work towards better understanding the mechanism and specificity of these enzymes, in the hopes of developing new tools to study A-to-I RNA editing. In the past our lab has extensively studied ADAR2, one member of this enzyme family. We have incorporated nucleoside analogues at the editing site to probe the active site, both before any structural information was available and afterwards to complement it. None of this was possible for ADAR1 until our recent characterization of a new ADAR1 substrate RNA, described in Chapter 2. Discovery and characterization of this editing site allowed us to develop an assay to probe the ADAR1 active site using nucleoside analogues. Chapter 3 details the development and use of this assay to uncover similarities and differences in how ADAR1 and ADAR2 recognize their substrate. These differences may pave the way for development of ADAR-specific inhibitors, and further use of this assay may allow us to uncover additional intriguing differences within this family of enzymes. With the abundance of new editing sites coming to light due to recent deep sequencing studies, more tools are needed to elucidate the biological consequences of these editing events. We developed substrate-specific inhibitors of editing by targeting RNA structure and sequence, described in Chapter 4. Importantly, we found that antisense oligonucleotides can bind to ADAR substrate RNAs, disrupt the native secondary structure and inhibit editing. We tested three different analogues and found that locked nucleic acid/2'-O-methyl mixmer oligonucleotides work most efficiently to inhibit editing. This will be an important new tool for the field, as labs can now use antisense oligonucleotides to inhibit editing of their RNA of choice. Finally, we developed several new assays for ADAR2 editing, for the most part based on the serotonin 2C receptor (5HT(2C)R) pre-mRNA. This work is described in Chapter 5. Similar assays have been used in the past with the GluR-B R/G site RNA, but adapting them to use the 5HT(2C)R RNA means that new sequence and secondary structure questions can now be addressed. In addition, we have used these assays to investigate how the part of ADAR2 linking the second double-stranded RNA binding domain and the catalytic domain may influence specificity and activity.