Author ORCID Identifier

Defense Date


Document Type


Degree Name

Doctor of Philosophy



First Advisor

Matthew Hartman

Second Advisor

Ashton Cropp

Third Advisor

Katharine Tibbetts

Fourth Advisor

Brian Fuglestad

Fifth Advisor

Aaron May


In an era of growing enthusiasm regarding peptide therapeutics, the ability to introduce unnatural amino acids allows optimization of chemical and physical properties, ultimately leading to more drug-like peptides. However unnatural amino acid delivery is a complicated process that frequently involves issues with efficiency and site selectivity. While the ribosome ultimately drives the chemical acceptance of an unnatural amino acid into a peptide, transfer RNA (tRNA) is responsible for the translocation of the unnatural amino acid to the ribosome, and the site-specific pairing to the proper codon triplet in the messenger RNA (mRNA). Thus, tRNA is an important and perhaps underappreciated variable in the efficient and selective translation of unnatural amino acids. This makes the source of tRNA, either cellular or synthetic, an interesting topic, as both have different properties due to the presence of chemical nucleotide modifications on cellular, but not synthetic tRNA.

Here I present a novel method for the isolation of modified tRNA from E. coli cells that pairs liquid-phase hybridization and fluorous affinity chromatography. Using this method, I was able to purify half of the E. coli tRNAs to homogeneity. By designing a competitive in vitro translation assay, I demonstrate that cellular tRNA captured by my method achieves more efficient codon readthrough, and thus amino acid delivery, than unmodified synthetic tRNA. However, in more complex codon competition assays involving as many as five competing tRNAs, I observe unexpected codon readthrough by cellular tRNAs. These breakdowns in specificity create a barrier that prevents the translation of homogeneous peptide products bearing non-canonical amino acids. By pairing mutant ribosomes with a hyper-accurate phenotype and synthetic tRNAs lacking key chemical modifications, I overcome this barrier and demonstrate the translation of homogeneous peptides bearing up to four non-canonical amino acids. My work presents an accessible method for isolation of individual cellular tRNAs, characterizes their strengths and weaknesses in an in vitro translation system, and shows that the synthetic tRNA – mutant ribosome pairing can be used to reassign the genetic code far beyond what has previously been demonstrated, allowing the translation of peptides bearing numerous non-canonical amino acids.


© Clinton Arritt Lloyd McFeely

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VCU Theses and Dissertations

Date of Submission


Available for download on Sunday, October 31, 2027