Date of Award
Ph.D. in Pharmaceutical Sciences
Kristine L Willett
David A. Colby
G-quadruplexes (G4s) are non-canonical secondary nucleic acid structures that exit in various biologically important regions within a cell, such as the promoters of DNA, 5’-Untranslated regions (UTR) of mRNA and telomeres. These secondary structures tend to cluster around 1 kb from transcriptional start sites across all human chromosomes in addition to at telomeric regions. Extensive efforts from many research groups have been dedicated to targeting G4s in oncogenic promoters, telomeres, and 5’-UTRs with traditional small molecules. However, most small molecules recognizing multiple G4s raising potential issues in terms of utilizing these small molecules clinically. Therefore, we developed a novel nucleic acid clamp (NA-clamp) based strategy, which takes advantage of both the highly specific complementary base pairing capability of nucleic acids and the length variation of nucleic acid structures, in order to stabilize individual G4s with high specificity. In this work, we explored the possibility of applying this NA-clamp approach on both transcriptional and translational levels. Specifically, we demonstrated this NA-clamp approach on MYC promoter G4 and NRAS 5’-UTR G4. Silencing MYC and NRAS expression has been demonstrated to be an effective approach to induce apoptosis of lymphoma and breast cancer, and melanoma, respectively. Targeting and stabilizing G4 structures formed in the promoter of MYC and 5’-UTR of NRAS has been shown effective at silencing their expression. In this body of work, we designed clamps specific for both the MYC promoter G4 and the NRAS 5’-UTR G4. we examined the binding specificity and G4 stabilizing capability of the designed clamps to their corresponding target using electromobility shift assay, electronic circular dichroism and DMS footprinting. Further studies include in vitro cytotoxicity, luciferase assay, fluorescent microscopy, ChIP-PCR, qPCR and western blot were used to evaluate the pharmacological effect of clamps on tumor cells. This research provided insights on the applicability of NA-clamps as specific G4 stabilizers that modulate gene expression in cancer cells, providing the foundation for the future development of NA-clamps into effective therapeutics, or as diagnostic companions, against many cancer types associated with MYC or NRAS.
Hao, Taisen, "Nucleic acid clamp-mediated transcriptional and translational modulation of oncogenes via the stabilization of G-quadruplexes" (2017). Electronic Theses and Dissertations. 1479.