In vivo bioluminescence imaging (BLI) is a powerful tool for visualizing disease progression, tracking gene expression, identifying new therapeutics, and more. BLI is useful for studying one cellular phenomenon at a time; however, using it to monitor several processes at once has been challenging due to its lack of distinguishable probes. To create tools for multi-component BLI, scientists have sought to create orthogonal mutant luciferase/modified luciferin pairs. Recently, the pair named “Cashew” and “Pecan” was developed by the Prescher Lab at UC Irvine through directed evolution. It outperformed other pairs in terms of brightness, stability, and orthogonality when tested in a mouse model. Here, we assess the specific amino acids that confer substrate selectivity on this leading orthogonal pair of luciferases. Singular point mutants of the parent enzymes were cloned, characterized and assessed for substrate selectivity. Mutations, such as G316S, were determined to diminish selectivity more than 10-fold; reversion of G316S improved selectivity by 23% without diminishing brightness. We also identified key mutations essential to positively conferring selectivity. With this knowledge, future engineering efforts can be better informed to maximize selectivity of luciferases. Ultimately, this can lead to improved sensitivity of in vivo multicomponent imaging.

Cloning and characterizing D-luciferin and 4-bromoluciferin selective mutant luciferases for in vivo bioluminescence imaging

2019 Fall National Meeting

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