Aequorin Mutants with Site-Specifically Incorporated Non-Natural Amino Acids for Biomedical Applications

Light emitting molecules are an indispensable part of detection and reporting in many fields and are employed in a variety of biomedical applications. Bioluminescent light, or living light, from bioluminescent proteins in particular has many beneficial characteristics, including their lack of a need for an outside excitation source and detection at as low as subattomole levels. Aequorin is a well-characterized bioluminescent photoprotein that has found application in in vitro and in vivo studies. Despite the many advantages of aequorin, its application has been limited by the finite number of canonical amino acids restricting the engineering of aequorin. In order to increase the applications of aequorin, we have taken established methods that hijack the cellular machinery used to synthesize proteins to incorporate non-natural amino acids. By site-specifically incorporating the non-natural amino acids L-4-aminophenylalanine, L-4-bromophenylalanine, L-4-iodophenylalanine, and L-4-methoxyphenylalanine, into positions associated with the bioluminescence and charging them with analogs of coelenterazine, several red-shifted aequorins, including the most red-shifted aequorin to date, with half-lives of up to 60 s were developed, creating aequorin mutants suitable for multiplexing and for transparent and deep tissue imaging. An additional non-natural amino acid, L-4-azidophenylalanine, for bio-orthogonal linking via click chemistry was incorporated at position 69 of aequorin. This form of orthogonal reaction can be reliably performed with any two molecules that contain the azide and alkyne reactive groups required for click reactions, even in complex samples, with no side reactions. The L-4- azidophenylalanine substituted aequorin was successfully covalently linked to a fluorophore via the azide to alkyne click reaction for BRET. Aequorin was also genetically linked to a VEGFA targeting molecule, a DARPin designated as MP0112, for the imaging of neovascularization in vivo using a wet age-related macular degeneration model in mice induced by laser exposure. By doing so, we developed the first bioluminescent imaging system using a DARPin to directly image VEGFA in the retina