Exploring the Limits of Ribosome Mediated Polymerizations for Expanding the Genetic Code

PROJECT SUMMARY Recombinant protein production (RPP) has become a powerful tool for producing life-saving therapeutics such as insulin, monoclonal antibodies, and other critical biopharmaceuticals.

However, this promising technology is severely limited by the ability to efficiently expand the genetic code to incorporate exotic monomers and backbones for enhanced therapeutic function.

The vast majority of biopolymers currently produced by the translation machinery display polyamide backbones; therefore, the possible secondary and tertiary confirmations available to proteomimetics synthesized via RPP are limited to these scaffolds.

Since monomer sequence defines structure and structure defines function, expanding the available monomer pool for translation will produce biopolymers with greater structural complexity and thus increase the functional capabilities for proteomimetic therapeutics.

A major limitation to addressing this issue is the underexplored capability of the ribosome to incorporate unnatural monomers, especially those that do not form peptide (amide) bonds.

Toward this goal, this proposal aims to develop genetically encoded chemistries that can be catalyzed by the ribosome to synthesize sequence defined polymers (SDPs) with structurally diverse backbones (non-peptide bonds).

Specifically, I will design and synthesize a library of a-hydrazino-keto ester monomers, charge them onto orthogonal tRNA, introduce them to the translation machinery in vitro, and evaluate the ability of the ribosome to catalyze their polymerizations. The hydrazine and keto ester moieties are known to react in solution to form various heterocyclic products.

Importantly, the last mechanistic step in heterocyclic formation is amide bond formation, a specialty of the ribosome.

Therefore, I hypothesize that bifunctional monomers comprised of both these moieties are capable of forming heterocyclic linkages via ribosome mediated catalysis.

Encouraging preliminary results by our collaborative and interdisciplinary research team have suggested this goal is achievable as we have found the ribosome to be more accommodating than previously thought.

The experiments in this proposal will (1) broaden our understanding of molecular translation, (2) elucidate the limitations and principles that govern genetic code reprogramming, and (3) expand the synthetic toolbox for the development of biologically derived SDPs via the ribosome.

Accomplishing the aims in this proposal will increase the backbone diversity currently attainable by the translation machinery and could produce SDPs that might exhibit greater therapeutic efficacy.