DOI
https://doi.org/10.25772/EVK4-1K18
Defense Date
2023
Document Type
Thesis
Degree Name
Master of Science
Department
Chemistry
First Advisor
Matthew Hartman
Abstract
The ability to create diverse macrocyclic libraries containing non-canonical amino acids (ncAAs) for drug discovery has been enabled through advances in understanding the plasticity of the translation apparatus towards ncAAs. These ncAA incorporation experiments have been enabled by the incredible control of the translation system offered by reconstituted cell free translation systems. Typical experiments testing for ncAA incorporation involve radioactive endpoint assays to measure yield alongside mass spectrometry experiments to validate incorporation. These endpoint assays require significant post-experimental manipulation for analysis, and prevent higher-throughput analysis and optimization experiments. Continuous assays for in vitro translation all involve the synthesis of fluorescent proteins which require the full complement of canonical AAs for function, and are therefore of limited utility for testing of ncAAs. Here we describe a new, continuous fluorescence assay for in vitro translation based on detection of a short peptide tag using an affinity clamp protein which changes its fluorescence emission ratio signal upon binding. Using this assay in 384 well format, we were able to validate the incorporation of multiple ncAAs and also quickly test for the codon reading specificities of a variety of E. coli tRNAs. With continuous, safer, high-throughput translation methods available, advancements in drug therapy are becoming boundless.
Rights
© Gianna Kerestesy
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-12-2023