Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Chemistry, Biochemistry

Date of Defense

4-18-2017

Graduate Advisor

James K. Bashkin

Committee

Benjamin J. Bythell

Keith J. Stine

Chung F. Wong

Abstract

Pyrrole-imidazole polyamides (PAs) represent a class of synthetic ligands that bind to the minor groove of DNA through hydrogen bonds and can be programmed to recognize predetermined DNA sequences. Potential applications of these compounds include gene expression modulation, antimicrobial activity and cell imaging.

Studies conducted in our lab in collaboration with NanoVir have shown that large PAs exhibit antiviral activity against Human Papillomavirus (HPV), which is the most common sexually transmitted infectious agent in the US and the primary factor for the development of cervical cancer. PAs act against HPV by targeting the viral DNA and promoting its selective elimination from cells with a mechanism involving the DNA Damage Response (DDR) pathway, although the details underlining this mechanism are still under investigation. In a library of more than 100 PAs, we observed that even minor changes in polyamide chemical structure can cause dramatic differences in antiviral activity.

The work presented in this thesis focuses on exploring some of the factors that may play a role in determining the different effectiveness of compounds that are structurally similar. The first part of this report describes the work intended to assess the interactions occurring between pyrrole-imidazole polyamides and HPV-DNA fragments. The DNA fragments used in these studies correspond to the Long Control Region of HPV, a regulatory region of primary importance for viral replication and transcription. The preferred binding sites as well as the strength of the binding were investigated ii through DNase I footprinting and affinity cleavage coupled with capillary electrophoresis. This work provided information on a molecular level on the binding mode of large PAs with a viral DNA sequence, showing interesting differences in comparison to the shorter PAs usually described in the literature and showing how minor structural substitutions can change the binding mode of these compounds when interacting with DNA. The second part of this report focuses on the investigation of cellular uptake of pyrrole-imidazole polyamides in human cells. HPLC and LC-MS/MS methods were developed to assess cell-associated polyamide. The methods were employed to explore the permeability of polyamides differing in their N-terminal groups or polyamides paired with different counterions.

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