UC Davis

A promising therapeutic to reverse disease-causing point mutations at the RNA level is Adenosine Deaminases acting on RNA (ADARs). By recruiting endogenous ADARs to a therapeutically relevant location in the transcriptome, Adenosine (A) can be hydrolytically deaminated to Inosine (I), which is read as Guanosine (G) by translation machinery because of its Watson-Crick-Franklin hydrogen bonding face. Disease causing point mutations could therefore be reversed at the RNA level. This process would take advantage of ADARs’ exclusive editing of double stranded RNA by introducing an exogenous guide RNA (gRNA) that is complementary to the target site, thereby recruiting endogenous ADARs. To increase the efficiency of the ADAR reaction, to resist nuclease degradation, and to increase delivery, chemical modifications can be introduced to the gRNA. Each chemical modification at each position of the oligonucleotide has the potential to affect the efficacy of the ADAR reaction.

The overall goal of the majority of my work is to interrogate the effect of individual chemical modifications in gRNAs recruiting ADARs. In Chapters 2 and 3, this is done in vitro, and in Chapter 4, this is done in cells. The focus of much of my research is on the -1 position—the position of the gRNA that is directly opposite the base 5’ to the editing site. This position adopts a unique conformation in crystal structures of ADAR2 bound to double stranded RNA. Chemical modifications which either promote or prevent this conformation from occurring are placed at the -1 position of a 5’-UA (Chapter 2) or 5’-AA (Chapter 3) site. I have found for both sequence contexts that chemical modifications which promote the conformation observed at the -1 position (ie. 2’-deoxy nucleotides) enhance ADAR editing rate, while modifications which prevent the conformation from occurring (ie. locked nucleic acids) can abolish ADAR editing.

In Chapter 2, I also look at the combined effects of chemical modifications at the -1 and orphan positions, where the orphan position is the nucleotide of the gRNA opposite the target adenosine. In Chapter 3, I study a target which has many adenosines that are ADAR targets, allowing nucleoside analogs initially intended to be investigated as -1 analogs to be investigated as at other positions (ie. orphan, +2, and -3).

In Chapter 3, I begin by discussing cellular assays for measuring ADAR editing rate and my optimization of one particular cellular ADAR editing assay—the dual luciferase assay. I then apply this assay to -1 analogs at a 5’-UA target, confirming trends observed in vitro. I then show that endogenous ADARs can be recruited to the target site and measured by both the dual luciferase assay and next generation sequencing (NGS).

Finally in Chapter 4, I discuss insights into pedagogy gained during teaching experiences as a UC Davis graduate student. I present the data from analysis of a general chemistry co-course for underrepresented minority students. We find that participation in this co-course, which includes both academic, social, and meta-cognition support, decreases the grade gap observed between students in our class and their peers, and show student reported success of the program in achieving its social and meta-cognition goals. I then discuss insights gained from participation in the Future Undergraduate Science Education (FUSE) program, including formal training in pedagogy and as an instructor of record.