Hili, RyanChung Kim Chung, Kimberley Alexandra2025-04-102025-04-102024-11-182025-04-10https://hdl.handle.net/10315/42802In recent years, RNA-modifying enzymes have gained significant attention due to their impact on critical RNA-based processes, and consequently human pathology. However, identifying sites of modifications throughout the cellular transcriptome remains challenging largely due to the lack of accurate and sensitive detection technologies. Indeed, N2-methylation at guanosine such as N2-methylated guanosine (m2G) and N2,N2-dimethylated guanosine (m22G) are prevalent modifications found in RNA but their functional studies are limited due to the absence of sequencing methods. While m22G creates termination during reverse transcription and can often be detected during sequencing due to its high error signature, m2G does not affect Watson−Crick−Franklin base pairing, and therefore cannot be distinguished from unmodified guanosine. Although computational predictors exist for m2G, there are no direct sequencing method available. In this thesis, the first chemistry-based sequencing method for N2-methylated guanosine at the single-nucleotide level is described. This method takes advantage of the chemoselectivity of photoredox chemistry to enable selective photo-oxidation of m2G and m22G in the presence of guanosine. Under blue light, using riboflavin as a photocatalyst and selectfluor as an oxidant, N2-methylated guanosines convert into their respective 2,5-diamino-4H-imidazol-4-one (Iz) and 8-oxoguanine (OG) products resulting in different mutation signature after sequencing. The limitation and scope of this method were further evaluated and extended to explore other methylated guanosine types such as m1G and generate the first database of high confidence m2G sites in multiple RNA types in human lung cancer cell line.Author owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.Electronic Thesis or Dissertation2025-04-10m2Gm22GPost-transcriptional modificationRNA sequencingPhoto redoxEpitranscriptomicsTHUMPD3