J. Am. Chem. Soc. 139, 2351–2358 (2017)

Credit: JACS

Bioluminescence imaging techniques use luciferase to catalyze the oxidation of a luciferin substrate, producing light. While natural and artificial variants of both enzyme and substrate enable imaging with different wavelengths of light, the poor selectivity of luciferases for their cognate luciferins (over other structurally similar luciferins) hampers their use in multicomponent imaging. To address this shortcoming, Jones et al. targeted both sides of the luciferase–luciferin pair to engineer orthogonality. First, the authors designed and synthesized a panel of luciferin analogs with increased steric bulk that weakened their binding to the wild-type luciferase while retaining their intrinsic potential for light emission. The authors then generated libraries of luciferase variants by targeted mutagenesis at residues predicted to be important for substrate binding. Screening this mutant library for enzymes that were active with luciferin analogs also enabled iterative rounds of library diversification, and a secondary screen rated all of the pairwise combinations of enzyme and substrate for orthogonality. From these screens, three luciferases were identified that were capable of orthogonal activation by select luciferin analogs in vitro and in cell culture. These new luciferase–luciferin pairs are themselves potentially useful contributions to the bioluminescence toolbox, and this approach to their generation could be applied to future efforts in developing selective imaging probes.