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Title: Molecular and cellular evaluation of novel DNA sequence selective polyamides
Author: Lin, S.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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Pyrrole (P)-Imidazole (I) containing polyamides are small synthetic molecules which can target predetermined DNA sequences with high affinity and modulate gene expression by interfering with the binding of transcription factors to DNA. Hairpin and H-pin analogues show enhanced DNA binding affinity and selectivity compared to monomers, but are associated with poor cellular uptake. To address this problem, a new class of unlinked polyamide precursors containing appropriately designed pendant reactive and biocompatible functional groups is presented in this thesis. The ability of these agents to interact in situ and potentially form either H-pin or Hairpin conjugates was tested with DNase I footprinting analysis. The effects of modification on the formamido-imidazole-pyrrole-imidazole (f-IPI) polyamide and related analogues, by the addition of an amino group, on DNA binding affinity and sequence selectivity were also evaluated. Dbf4 is the regulatory subunit of Cdc7 kinase, which is essential for the initiation of DNA replication. Human Cdc7/Dbf4 (HuCdc7/Dbf4) kinase activity is critical for cell proliferation and high expression levels occur in multiple solid human malignancies. F-IPI can target the MIuI Cell cycle Box (MCB) sequence 5’-ACGCGT-3’which is the critical site for transcription factor binding and activation of expression of the HuCdc7/Dbf4 core gene in mammalian cells. DNA sequence selective binding of f-IPI to the MCB sequence was shown in DNase I footrpinting experiments. A f-IPI-induced, dose-dependent inhibition of protein binding to the MCB was demonstrated in an Electrophoretic Mobility Shift Assay (EMSA). RT-PCR and immunoblotting analysis confirmed the inhibitory effects of f-IPI on HuDbf4 in MDA-MB231 cells. Small interfering RNA-mediated depletion of HuDbf4 decreased cell survival and proliferation, and induced G1 arrest in MDA-MB231 cells. F-IPI treatments also showed a dose-dependent reduction of cell survival and proliferation and induced G1 arrest. Although the f-IPI H-pin analogue exhibited better DNA binding affinity and protein inhibition, no marked effects at mRNA and protein levels were detected in cells, likely attributed to its large size. The unlinked f-IPI analogues containing reactive groups [i.e f-IP(C3NH2)I and f-IP(C3Cl)I] able to interact in situ and form H-pin were tested in the Dbf4 promoter model system. f-IP(C3NH2)I presented the same DNA binding affinity and protein inhibition to the MCB as the combination of f-IP(C3NH2)I and f-IP(C3Cl)I (potential hybrid) but it produced much higher effects than f-IP(C3Cl)I. RT-PCR and immunoblotting analysis showed that the potential hybrid produced greater reduction of HuDbf4 mRNA and protein levels than either f-IP(C3NH2)I or f-IP(C3Cl)I. In addition, the potential hybrid demonstrated greater G1 arrest than each polyamide alone. Finally, a series of fluorescent f-IPI analogues are presented, providing an intrinsic probe for monitoring the cellular uptake of polyamides. Aza-Hx-PI was shown to target the same sequence as f-IPI (DNase I footprinting), inhibit protein binding (EMSA) and reduce the levels of HuDbf4 in cells. Time and dose-dependent nuclear localisation was detected by confocal microscopy. Overall the data presented in this thesis highlight the potential of small molecule polyamides as modulators of gene expression.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available