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Title: Controlled manipulation of gene expression with sequence-selective DNA-binding drugs
Author: Ellis, T.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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Three sets of synthetic DNA-binding drugs have been evaluated, largely by DNase I footprinting, to determine their affinity and selectivity for DNA sequences. MOLRAC bis-intercalator compounds produced by novel synthesis were found to be relatively insoluble with low affinity for DNA and little sequence-selectivity. Pyrrolobenzodiazepine (PBD) conjugates designed to bind covalently to DNA were shown to have reasonable affinity for DNA and moderate sequence-selectivity. Bacterial in vitro transcription assays demonstrated that covalent linkage to DNA could selectively block transcription at targeted sequences. Hairpin polyamides designed to bind reversibly within the minor groove were confirmed to have excellent affinity for DNA, remarkable sequence-selectivity, and the ability to modulate expression from gene promoters in vitro. Subsequently, the properties of hairpin polyamides were assessed in vivo. Fluorescent polyamide uptake by cultured cells was demonstrated using confocal microscopy. An assay was developed whereby HeLa cells were transfected with a reporter plasmid and then treated with polyamides targeted to the plasmid promoter. This showed that polyamides capable of inhibiting transcription in vitro were also capable of inhibiting gene expression in transfected cell culture. Expression from a promoter controlling the type 1 inositol-1,4,5-trisphosphate receptor (IP3R1) was also inhibited in transfected cells, this time using a combination of hairpin polyamides to increase specificity. The same combination could not, however, modulate endogenous gene expression in untransfected cells. In conclusion it is possible to manipulate gene expression in transfected cell culture in a controlled manner using DNA-binding drugs. The PBD conjugates may yield successful drugs with further development and their ability to halt transcription is promising. Hairpin polyamides have immediate potential as therapeutic and research tools.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available