Event | Nucleic Acids Seminar with Byron Purse
Fluorescent Nucleoside Analogues for Live-Cell Imaging of RNA Biology and Single-Molecule Studies
This Wednesday, March 18 at 11:00am-12:00pm, the Department of Chemistry is hosting a seminar on Nucleic Acids with .
Abstract |聽Fluorescent nucleoside analogues have emerged as powerful molecular probes for exploring RNA biology. Nucleobase analogue fluorophores that preserve Watson鈥揅rick pairing offer a distinct advantage: they can be seamlessly incorporated into nucleic acids with minimal perturbation to native structures and functions, while enabling precise investigations of conformation, dynamics, and interactions with proteins. In this work, we developed a suite of fluorescent nucleoside analogues with two main goals: (i) establishing metabolic labeling approaches for live-cell RNA imaging without cell fixation and staining, and (ii) achieving single-molecule detection nucleic acids labeled only with fluorescent nucleobases. For cellular imaging, we focused on ribonucleoside analogues recognized by uridine-cytidine kinase 2 (UCK2) in HeLa cells. We show that tC and pyrroloC are taken up by living cells, phosphorylated through the pyrimidine salvage pathway, and incorporated into RNA, enabling visualization of RNA synthesis, localization, and turnover.
Using confocal microscopy colocalization studies with mCherry-tagged stress granule protein G3BP1, P-body protein DCP1A, and RNA helicase DDX6, as well as selective RNA synthesis inhibitors, we observed rRNA enrichment in previously uncharacterized cytosolic stress granules associated with DDX6鈥攕tructures distinct from canonical stress granules and P-bodies. This labeling strategy is also compatible with nucleolar FRAP analysis, providing quantitative insights that support a viscoelastic model of the nucleolus, where proteins diffuse rapidly through an RNA-dense network. In parallel, drawing inspiration from xanthene dyes, we designed and synthesized ABN, a bright tricyclic nucleobase analogue detectable at the single-molecule level in oligonucleotides using smTIRF and fluorescence correlation spectroscopy. Collectively, these developments introduce versatile new tools for probing RNA and DNA behavior from the single-molecule to the cellular scale.
Speaker
Byron Purse grew up in Regina, Saskatchewan, Canada, and studied Chemistry and Biochemistry at the University of Regina. His interest in organic chemistry began during an undergraduate research internship with Prof. David Bundle at the University of Alberta, which led to his first publication. He completed his PhD at The Scripps Research Institute with Prof. Julius Rebek studying molecular recognition and reactivity in confined environments, followed by postdoctoral work with Prof. Jan-Erling B盲ckvall at Stockholm University on aerobic oxidation catalysis.