| The theme of this dissertation research centers on the design, characterization and development of ligands to target protein and RNA biomolecules. The focus is on the PDZ domain protein subfamily---specifically the third PDZ domain (PDZ3) of PSD-95---and the A-site RNA of the E. coli ribosome. The objective in both cases was to prepare ligands that could bind in a noncovalent manner, with high affinity and selectivity, each biomolecule. From this, it was intended that a basic understanding could be obtained regarding the molecular recognition properties for each system, and to then employ these ligands as cellular probes for biological investigations. Ultimately, successful ligand candidates could then potentially serve as lead compounds for therapeutic agent development.; For the first and larger portion of this dissertation comprising the PDZ domain studies, a variety of experimental techniques were implemented to examine different aspects of ligand development. T7 phage display was used to 253 identify binding sequences, several of which were used to prepare peptides for isothermal titration calorimetry experiments and PDZ3. T7 phage were also utilized in display cloning, wherein a brain-derived cDNA library was screened against several peptide-based ligands that had previously been shown to bind PDZ3. The T7 phage approach was the basis for the construction of an ELISA, which allowed for a rapid assessment of binding selectivity when using a small library of spatially-segregated clones derived from the PDZ1-2 dual domain and PDZ3 domains of four MAGUKs (PSD-95, SAP97, PSD-93 and SAP102). Finally, a specialized series of PDZ3-binding peptides was prepared in which the penultimate position of the C-terminus was modified with a variety of halogenated phenylalanine residues. In this manner, the influence of fluoro, chloro, bromo and iodo substitution could be evaluated for effect upon the binding thermodynamics.; The second portion of this dissertation involves the use of T7 phage to discover peptide sequences capable of binding the A-site RNA of the bacterial ribosome. Peptides obtained in such a manner were intended to possess ribosomal function disruption properties, and thus serving as possible new antibiotic agents or leads for further development. In addition to obtaining sequences for peptide preparation, this study also identified conditions for use in the various biopanning stages that could be used in future phage experiments against other RNA targets. |
