Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Christine S. Chow

Abstract

ABSTRACT

THE DEVELOPMENT OF PEPTIDE LIGANDS TO TARGET H69

by

DANIELLE NICOLE DREMANN

December 2015

Advisor: Prof. Christine S. Chow

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

In the development of peptide ligands to target H69, SPPS and ESI MS was used to determine if 1) peptides could bind to modified H69 and 2) if increased affinity for the target RNA could be enhanced with modification. An alanine and arginine scan was synthesized and tested for this determination. Selected peptides were then tested using biophysical techniques such as circular dichroism and isothermal titration calorimetry. An assay was also designed to increase the efficiency of peptide ligand selection in which novel peptides with high affinity and selectivity could be identified for future projects. This assay was done using flow cytometry, instrumentation capable of identifying beads, bound to the target RNA conjugated to a fluorophore, based on fluorophore emission, and sorting them into 96-well plates for MS analysis.

The last part of the research focused on aminoglycoside-H69 RNA interactions. ESI MS was used to obtain binding affinities and stoichiometries of 2AP- and A-containing H69 RNAs. The findings revealed that the binding mode had not changed between these two sets of RNAs, which revealed the value for using ESI MS in combination with other techniques, such as fluorescence, to give a complete picture of the binding mode (stoichiometry, affinity, selectivity) in comparison with conformational changes that may occur upon binding. Further exploration of aminoglycoside-H69 RNA interactions took place with the H69 peptide NQVANHQ-NH2 to determine whether a fragment-based drug design approach could be used to create small compounds for future in vivo applications.

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