UC Davis

Oligonucleotides are short polymeric chains of nucleic acids that are immensely useful for a plethora of scientific applications. By way of chemical modification, post-transcriptional processes such as RNA interference (RNAi) or RNA editing can be studied at the molecular level using oligonucleotides. More so, oligonucleotides, which typically use the simple mechanism of base pairing to a complementary target nucleic acid sequence of interest, can be exploited to create therapeutics against diseases. Researchers developing oligonucleotide-based therapeutics often face challenges of specificity, off-targeting effects, oligonucleotide trafficking, metabolic stability, cellular uptake, delivery to specific tissues, and more. Therefore, chemical modifications along the oligonucleotide sugar and phosphate backbone and, less commonly, the nucleobase are used to overcome these obstacles.

In Professor Peter Beal’s lab, we take particular interest in designing nucleobase modifications that can be incorporated onto oligonucleotides to further understand the structure and function of RNA that can bind to other RNA or RNA-binding proteins in RNAi or RNA editing. This dissertation will describe chemically modified oligonucleotides that were used to study their effects on the catalytically active RNAi protein, Argonaute2 (Ago2), and the adenosine-to-inosine (A-to-I) editing enzyme adenosine deaminase acting on RNA (ADAR). Chapters 2 and 3 describe a published study in examining the effects of chemically modified antisense oligonucleotides targeting microRNA (anti-miRs) at the 3’-end nucleotide that can interact with a nucleotide-binding pocket in human Ago2 (hAgo2) when loaded with a microRNA strand. First, Chapter 2 describes the earlier stages of this work and goes into detail a molecular docking method to computationally screen 1’-triazole modified nucleotide analogs that can interact with the hAgo2 pocket. Additionally, precursor anti-miRs with a 3’-end, 1’-alkynyl modification were designed and tested in an established cellular based assay to confirm if the anti-miR could be converted into triazoles with various substituents using copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) reactions. We docked a library of 190 triazole-modified nucleotides, carefully evaluated the scoring of each docked ligand, and picked 17 triazoles that could be modified onto a 2’-O-methylated 15mer anti-miR with 1’-alkynyl 3’-end nucleotide. Chapter 3 continues to describe the identification of a significantly potent ester triazole-modified anti-miR among the 17 triazoles screened. Numerous investigative experiments were then conducted to determine the mechanism by which the ester modification elicits high potency. Such experiments included structure activity relationships, generalizability to two anti-miR sequences, nuclease resistance, miRNA target de-repression analyses, and a biotinylated anti-miR pulldown assay to determine hAgo2 binding affinity of the ester-modified anti-miR. Finally, Chapter 4 describes preliminary work to evaluate inhibition of ADAR1 in cultured cells using either an 8-azanebularine-containing RNA duplex inhibitor or ADAR mutant proteins that can impede dimerization needed for A-to-I editing activity.