UBC Theses and Dissertations
In vivo detection of G-quadruplex nucleic acids using monoclonal antibodies Henderson, Alexander Harper Hewitt
G-quadruplex nucleic acids are a group of nucleic acids formed from the non-Watson-Crick base pairing of guanine nucleic acids. They can readily form at physiological pH and physiological temperatures within sufficiently long stretches of guanine-rich oligonucleotides. Although, the existence of the G-quartet (the fundamental unit of a G-quadruplex) in a Petri dish has been recognized since the early 60’s, the existence of G-quadruplex nucleic acids in mammalian cells remains unclear. Yet while unequivocal evidence of the existence of G-quadruplex nucleic acids in live cells remains unclear, interest in these potentially important biological structures continues to intensify. G-quadruplex nucleic acids have been suggested to play key roles in essential human molecular pathways including telomere biology, transcriptional regulation and disease development. One of the major obstacles in G-quadruplex nucleic acid research is a lack of tools for the in vivo detection of these structures. In our work, we have harnessed hybridoma technology to produce the first monoclonal antibodies to these unique nucleic acid structures. To our knowledge, these are the first hybridomas secreting monoclonal antibodies obtained through the immunization of mice with purified and validated G-quadruplex structures. Monoclonal antibodies have been approved for use in diagnostic tests and for therapeutic treatments in both cancer and autoimmune diseases, and continue to be very effective laboratory research tools. Using monoclonal antibodies to different G-quadruplex nucleic acids we have explored the existence of G-quadruplex nucleic acids in mammalian cells. One of our antibodies, termed 1H6, forms discrete nuclear foci in human and murine cells and strong nuclear staining in most cells of human tissues. Based on the specificity of the antibodies for defined G-quadruplex structures in vitro, these foci could represent the detection of G-quadruplex nucleic acid structures in mammalian cells. If so, the work presented here provides the first direct evidence for the existence of G-quadruplex nucleic acid structures in human cells.
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