DOI
https://doi.org/10.25772/0FC2-SH29
Author ORCID Identifier
https://orcid.org/0000-0002-5712-1060
Defense Date
2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
Matthew Hartman
Second Advisor
Joshua Sieber
Third Advisor
Brian Fuglestad
Fourth Advisor
Sally Hunnicutt
Fifth Advisor
Keith Ellis
Abstract
In an era witnessing a surge in the popularity of peptide therapeutics, the incorporation of non-canonical amino acids (ncAA) emerges as a promising avenue for optimizing chemical and physical properties. While leveraging natural biomachinery facilitates the utilization of high-throughput peptide library screening methods, the implementation of a system receptive to ncAA presents inherent complexities. The seamless operation of this biomachinery necessitates the ribosome's acceptance of transfer ribonucleic acid (tRNA), wherein tRNA assumes the crucial role of delivering amino acids (AA) through non-covalent base pair interactions. Consequently, modifying ribonucleic acid (RNA) becomes imperative to efficiently guide the codon-anticodon interaction.
In this context, I introduce a significant advancement in the realm of unnatural base pairs (UBP), aimed at expanding the genetic code. This advancement commences with the optimization of the three-part building block unnatural nucleoside triphosphate (NTP), addressing key aspects in the nucleobase, sugar, and triphosphate components. Subsequently, I showcase the optimization of UBP incorporation through in vitro transcription (IVT) and employ strategic analytical methods to assess RNA purity. Finally, I evaluate the performance of UBP in the translation system, investigating their orthogonality both with and without the introduction of UBP-tRNA. The culmination of my work demonstrates the successful acceptance of a specific UBP sequence in the codon-anticodon interaction, utilizing E. coli Wild-type (WT) ribosomes.
Rights
© Anthony V Le
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-14-2023