Programmable Genetic Manipulation of Protein-Protein Interactions

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Randall Hughes, Arti Pothukuchy, Andre Maranhao, and Andrew D. Ellington

The Applied Research Laboratories, United States

As the engineerable properties of biomolecules and the synthetic networks created from them are elucidated, the augmentation of natural functionalities will become increasingly important to create ever complex and increasingly abiotic enzymes and pathways. One such instantiation is to introduce synthetic building blocks (nucleotides, amino acids) into biomolecules to augment or completely redefine their function and interactions with other biomolecules. The programmable nature of nucleic acids, with their defined interactions between complementary nucleobases has made them an attractive medium for the creation of biologically-inspired materials such as DNA hydrogels, DNA origami nanostructures, etc…. Unlike nucleic acids, protein-protein interactions are not based on a straightforward and thus readily engineerable interaction rule set. Instead interacting proteins have evolved idiosyncratically to interact with one another based on the ‘drunken-walk’ of natural selection. The ability to control the interaction and localization of protein-protein interactions in a predictable manner, especially proteins introduced from exogenous sources, could help to improve flux through synthetic metabolic pathways, enable the formation of novel protein chimeras as well as enable the directed evolution of interacting protein pairs. However, to date progress in this vein has been tepid. Herein we present our progress towards the genetic incorporation of nucleobase functionalized amino acids into proteins. These novel, synthetic amino acids contain the standard L-amino acid backbone with sidechains that include the nucleobase functionalities from nucleic acids. When placed in the proper structural context within a protein sequence, these amino acids should allow for programmable interactions between proteins similar to the strands of a DNA molecule. To facilitate the genetic incorporation of nucleobase amino acids, we have developed novel orthogonal aminoacyl tRNA synthetase/tRNA pairs as scaffolds for the directed evolution of amino acid specificity. Additionally, we report on the augmentation of these orthogonal pairs for incorporation of nucleobase and other unnatural amino acids.