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Scholarly Works (3 results)

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UC Davis Electronic Theses and Dissertations (2023)

Adenosine Deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in duplex RNA. The inosine product preferentially base pairs with cytidine resulting in an effective A-to-G edit in RNA. ADAR editing can result in a recoding event alongside other alterations to RNA function. A consequence of ADARs’ selective activity on duplex RNA is that guide RNAs (gRNAs) can be designed to target an adenosine of interest and promote a desired recoding event. One of ADAR’s main limitations is its preference to edit adenosines with specific 5’ and 3’ nearest neighbor nucleotides (e.g. 5’U, 3’G). Current rational design approaches are well-suited for this ideal sequence context but limited when applied to difficult-to-edit sites. Another limitation of ADAR editing is its specificity. ADARs can edit non-target sites due to the presence of good targets elsewhere. To achieve more specific editing, a gRNA may be discovered that allows for a reduction in off-target editing.In this dissertation, I describe a strategy for the in vitro evaluation of very large libraries of ADAR substrates, called En Mass Evaluation of RNA Guides (EMERGe). EMERGe allows for a comprehensive screening of ADAR substrate RNAs that complements current design approaches. I used this approach to identify sequence motifs for gRNAs that enable editing in otherwise difficult-to-edit target sites. EMERGe provides an advancement in screening that not only allows for novel gRNA design, but also furthers our understanding of ADARs’ specific RNA-protein interactions. Chapter 1 provides a general introduction to ADARs and their application for site-directed RNA editing. This chapter also describes current advancements in gRNA design and discovery. Chapter 2 outlines the studies used before the EMERGe assay. Additionally, this chapter will also describe the development of the EMERGe assay. The validation of the EMERGe assay is discussed within Chapter 3. Other discoveries made by the EMERGe assays will be tested in vitro within this chapter. Finally, Chapter 4 discusses in cellula applications of EMERGe-derived results. This includes testing EMERGe-derived gRNAs, in cellula results with a previously untested chemical modification, and the limitations of a new yeast-based assay.

    Cover page: Development and Application of Oligonucleotide Assays for Site-Directed RNA Editing
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2023)
    Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in duplex RNA. The inosine product preferentially base pairs with cytidine resulting in an effective A-to-G edit in RNA. ADAR editing can result in a recoding event alongside other alterations to RNA function. A consequence of ADARs' selective activity on duplex RNA is that guide RNAs (gRNAs) can be designed to target an adenosine of interest and promote a desired recoding event. One of ADAR's main limitations is its preference to edit adenosines with specific 5' and 3' nearest neighbor nucleotides (e.g., 5' U, 3' G). Current rational design approaches are well-suited for this ideal sequence context, but limited when applied to difficult-to-edit sites. Here we describe a strategy for the in vitro evaluation of very large libraries of ADAR substrates (En Masse Evaluation of RNA Guides, EMERGe). EMERGe allows for a comprehensive screening of ADAR substrate RNAs that complements current design approaches. We used this approach to identify sequence motifs for gRNAs that enable editing in otherwise difficult-to-edit target sites. A guide RNA bearing one of these sequence motifs enabled the cellular repair of a premature termination codon arising from mutation of the MECP2 gene associated with Rett Syndrome. EMERGe provides an advancement in screening that not only allows for novel gRNA design, but also furthers our understanding of ADARs' specific RNA-protein interactions.
      Cover page: Library Screening Reveals Sequence Motifs That Enable ADAR2 Editing at Recalcitrant Sites
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2024)
      Adenosine Deaminases Acting on RNA (ADARs) are members of a family of RNA editing enzymes that catalyze the conversion of adenosine into inosine in double-stranded RNA (dsRNA). ADARs selective activity on dsRNA presents the ability to correct mutations at the transcriptome level using guiding oligonucleotides. However, this approach is limited by ADARs preference for specific sequence contexts to achieve efficient editing. Substrates with a guanosine adjacent to the target adenosine in the 5 direction (5-GA) are edited less efficiently compared to substrates with any other canonical nucleotides at this position. Previous studies showed that a G/purine mismatch at this position results in more efficient editing than a canonical G/C pair. Herein, we investigate a series of modified oligonucleotides containing purine or size-expanded nucleoside analogs on guide strands opposite the 5-G (-1 position). The results demonstrate that modified adenosine and inosine analogs enhance editing at 5-GA sites. Additionally, the inclusion of a size-expanded cytidine analog at this position improves editing over a control guide bearing cytidine. High-resolution crystal structures of ADAR:/RNA substrate complexes reveal the manner by which both inosine and size-expanded cytidine are capable of activating editing at 5-GA sites. Further modification of these altered guide sequences for metabolic stability in human cells demonstrates that the incorporation of specific purine analogs at the -1 position significantly improves editing at 5-GA sites.
        Cover page: Nucleoside Analogs in ADAR Guide Strands Enable Editing at 5-GA Sites.
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