Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Chemistry, Biochemistry

Date of Defense

4-12-2017

Graduate Advisor

James K. Bashkin, Ph.D.

Committee

Wesley R. Harris, Ph.D.

Chung F. Wong, Ph.D.

Bethany K. Zolman, Ph.D.

Abstract

Human papillomavirus is a small dsDNA virus that infects mucosal and cutaneous epithelial tissues. Persistent infection with high-risk HPV is the main etiological agent in the development of cervical cancer worldwide. Although prophylactic vaccines against HPV are available, these preventative measures are type-specific and are ineffective against existing infections. Thus, there is a pressing need for antiviral drugs with a broad-spectrum activity against HPV to eradicate existing infections, no matter the subtype.

Our group and collaborators have synthesized an extensive library of novel N-methylpyrrole/N-methylimidazole (Py/Im) hairpin polyamides (PAs) with broad-spectrum activities against three prevalent oncogenic-HPV types (HPV16, HPV18 and HPV31) without apparent cytotoxicity. Because Py/Im polyamides can be rationally designed to bind the minor groove of dsDNA in a sequence-dependent manner, these small molecules are attractive candidates as modulators of gene expression, as molecular probes for diagnostics and as antiviral agents for the selective elimination of dsDNA viruses.

Despite the promise of applying these agents to treat HPV infections, much regarding their mechanism of action remains unexplained. Therefore, the overall goal of this dissertation is to investigate the DNA-binding properties of potent anti-HPV PAs under cell-free conditions and in HPV-harboring keratinocytes. Chapter 2 describes biophysical studies of anti-HPV hairpin polyamides (PA1 and PA25) using deoxyribonuclease I footprinting and affinity cleavage to interrogate the sequence specificity, binding sites and dissociation constants within the HPV18 Long Control Region. Chapter 3 extends these analyses to a new structural class of hairpin polyamides with tetramethylguanidinium (PA30) and guanidinium (PA31) substitutions on the N-terminus of PA1. Chapter 4 presents a detailed procedure to determine the genome-wide binding events of DNA-binding molecules in cell-free conditions using hydroxyl radical footprinting coupled to massively parallel DNA sequencing (·OH-Seq). In Chapter 5, ·OH-Seq and COSMIC-Seq have been employed to assess the binding occupancies of anti-HPV hairpin polyamides across the viral genomes in keratinocytes harboring HPV16 episomes.

A deeper understanding of the molecular underpinnings of the ability of antiviral hairpin PAs to eliminate the HPV viral load may facilitate the development of a new generation of broad-spectrum antiviral treatments against this deadly virus.

Available for download on Friday, May 04, 2018

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