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Open Access Publications from the University of California

Scholarly Works (2 results)

  • Article
  • Peer Reviewed
UC Irvine Previously Published Works (2022)
Herein, we detail a novel reverse-transcription (RT) assay to directly detect chemical adducts on RNA. We optimize a fluorescence quenching assay to detect RT polymerization and employ our approach to detect N1-alkylation of inosine, an important post-transcriptional modification, using a phenylacrylamide as a model compound. We anticipate our approach can be expanded to identify novel reagents that form adducts with RNA and further explored to understand the relationship between RT processivity and natural post-transcriptional modifications in RNA.
    Cover page: A Fluorescent Reverse-Transcription Assay to Detect Chemical Adducts on RNA
    • Thesis
    • Peer Reviewed
    UC Irvine Electronic Theses and Dissertations (2024)

    Over the last few decades, the development and application of advanced fluorescent probes have revolutionized our ability to assess complex biological processes visually and quantitatively with unprecedented specificity. Employing these probes alongside sophisticated microscopy techniques, this dissertation explores the development and application of diverse technologies within biological research to enhance our understanding of RNA biology, drug interactions, and cellular mechanisms.

    The use of fluorescent probes in real-time metabolic labeling of RNA, both in cellulo and in vivo, is explored. This approach provides critical insights into RNA dynamics, enabling the precise measurement of RNA expression patterns that are pivotal for understanding cellular function in health and disease.

    The development of a fluorescent reverse-transcription assay for detecting covalent interactions between small molecules and RNA is detailed. This method offers valuable perspectives on RNA modifications and their potential implications in therapeutic development, particularly through the identification of small molecule binders that could lead to innovative treatment strategies. Novel photocrosslinking fluorescent probes were developed for a new spatial transcriptomics technology employing spatially restricted cell tagging by confocal microscopy and cellular isolation with fluorescence-activated cell sorting. This technique allows for high- resolution mapping of gene expression within specific cellular contexts, thus deepening our understanding of the spatial and temporal dynamics of gene activity crucial for comprehending complex biological phenomena.

    The exploration of drug permeability through the development of a new fluorescence cell-based assay highlights how this technology can assess the bioavailability of pharmacologically relevant molecules, particularly those that challenge the conventional boundaries of drug design. By enabling the direct measurement of compound accumulation in a cell-type independent manner, this method helps to redefine our approach to therapeutic development.

    Together, these investigations contribute new tools and methodologies to study intricate biological systems and their interactions with therapeutic agents, thereby offering valuable insights into the potential for diagnostic and therapeutic advancements.

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