Title

Genetically Encoded Biomaterials: Design, Characterization, and Applications for Functionalizing Biological Therapeutics

Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular, Cellular, and Developmental Biology

First Advisor

Isaacs, Farren

Abstract

Engineering of the translation apparatus has permitted site-specific incorporation of nonstandard amino acids (nsAAs) into proteins, thereby expanding the genetic code of organisms. Recent work in genome engineering has enabled whole genome recoding, in which codons removed from the genetic code can be repurposed as new sense codons dedicated for incorporation of nsAAs. This work describes our vision of how synthetic biology and genetic code expansion will allow the development of genetically encoded biomaterials -- a new class of biomaterials that marries the sequence-defined production, fidelity and processivity of ribosomal protein expression, with the vast molecular and structural diversity of the chemical world. We first chose the nsAA para-azido-phenylalanine (pAzF) as a particularly versatile tool for (multi-site) protein functionalization. The azide group in the side chain is a bioorthogonal chemical moiety that enables "click" reactions to attach diverse molecular structures. However, the azide moiety was found to be unstable in physiological conditions and pAzF is reduced to para-amino-phenylalanine (pAF). Azide reduction decreases yield of pAzF residues in proteins to 50-60% per residue, and thereby limits protein functionalization by click reactions. Here, we describe the use of a pH-tunable diazotransfer reaction that converts pAF to pAzF at >95% efficiency in proteins. The method selectively restores pAzF at multiple sites per protein while minimizing off-target modifications. This work addresses a key limitation in the production of pAzF-containing proteins for multi-site functionalization with diverse chemical moieties by restoring azides, setting the stage for the production of genetically encoded biomaterials.I We apply these technical advances to address the challenge of short half-lives of protein and peptide therapeutics in blood, which negatively impacts patient compliance and quality of life. Specifically, we show how multi-site incorporation of pAzF enables programmable, multi-site functionalization with fatty acids to generate a set of sequence-defined synthetic biopolymers with programmable binding affinity to albumin and, consequently, tunable serum half-lives spanning 5-94% of albumin's half-life in mice. This work enables the production of functionalized biotherapeutic proteins with programmable half-lives and establishes a technical foundation for the production of biopolymers where multi-site incorporation of chemical modifications at monomeric precision endow tunable chemical and biological properties. Finally, we describe our efforts to expand the genetic code with D-amino acids (DAA), which would enrich the complexity of accessible protein designs. Their incorporation in proteins can confer resistance to proteases, and make protein conformations accessible that otherwise could not be achieved. We describe our efforts to systematically identify and address the barriers to protein expression with Dtyr. Namely, efforts to enhance the concentration of intracellular Dtyr, to promote preferential charging of Dtyr, to limit the hydrolysis of the Dtyr-tRNA ester bond, and to lay the foundation for ribosome engineering to better facilitate translation with DAA. While this remains work in progress, we anticipate that the engineering efforts we describe will prove important to achieve the goal of incorporating DAA in cells. Collectively, we enable and demonstrate the utility of multi-site functionalization at pAzF residues, showing how new and tunable properties can be introduced in the protein or biopolymer. With efforts to further expand the genetic code withnonstandard amino acids (such as DAA), these ribosomal products will become more chemically distinct from natural protein products, truly becoming an independent class of programmable biomaterials with broad applications in medicine, materials science, and biotechnology.

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