Genetic Code Expansion: Maximal Sense Codon Reassignment in Pursuit of a 40-Amino Acid Genetic Code

Author

• Clark A. Jones, Virginia Commonwealth UniversityFollow

Author ORCID Identifier

https://orcid.org/0000-0002-6033-194X

Defense Date

2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Matthew Hartman

Second Advisor

Ashton Cropp

Third Advisor

Aaron May

Fourth Advisor

Soma Dhakal

Abstract

Biologic therapeutics have had a dramatic rise to popularity in recent years. As the tools available to chemical biology research expand, so do the possibilities. The chemical functionalities available for a given peptide are limited by the structures available to be encoded within it.  Due to this, research to induce encoding of unnatural, or non-canonical amino acids has been an area of great interest. High throughput screens such as mRNA display are capable of screening up to millions of randomized peptide structures in search of a potent binder. Efforts to expand the genetic code, enhancing the quantity of encodable amino acids, would significantly increase the quantity of available structures in a given screen. Given the strength of these screens is in the sheer quantity of potential peptides tested in a single screen, any method to increase this could dramatically increase the overall efficiency for these techniques. Additionally, the ability to introduce new structures and chemical motifs permit screens that can test for additional chemical functionalities, and permit the design of potent peptide binders.

Here, I present a novel design for breaking open the degeneracy of genetic sense codons in-vitro. I first utilize the flexizyme to optimize a method for efficiently aminoacylating non-canonical amino acids to select tRNAs. I’m then able to use these ncAA-tRNAs, in conjunction with our hyperaccurate ms12 ribosomes, to demonstrate a technique to develop selective codon-tRNA pairings into specific encoding of novel amino acids at a given codon. This permits the reassignment, and breaking open of the NCN serine, proline, and threonine codon boxes. Discoveries in the importance of optimal free Mg²⁺ ion concentration in translation reactions then permits the combined sense codon reassignment of these three codon boxes together, developing a 26-amino acid genetic code. I then further develop this method, applying this strategy across the genetic code to reassign the leucine, isoleucine, valine, methionine, arginine, and glycine codon boxes. These sense codon reassignments, in combination, are able to permit the formation of a 40-amino acid genetic code. This expansion can permit the utilization of expanded mRNA display screens, dramatically increasing the chemical potential of a given screen, and permitting the discovery of novel peptide therapeutics during the drug discovery stage. This flexibility of the genetic code as well allows researchers to tailor the composition of their genetic code for an mRNA display screen, optimizing the library to be predisposed to output strong protein binders.

Rights

© Clark Alexander Jones

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-8-2026