Firefly Luciferase Mutants Allow Substrate‐Selective Bioluminescence Imaging in the Mouse Brain

Abstract

Bioluminescence imaging is a powerful approach to visualize specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Here we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. Using adeno-associated viral (AAV) vectors to express luciferases in the brain, we find that mutant luciferases that are inactive or weakly active with D-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.

Orthogonal Luciferase–Luciferin Pairs for Bioluminescence Imaging

“…Fluc is a highly dynamic enzyme, 31,56 complicating the selection of residues from static structural or sequence data. Moreover, amino acids known to play key roles in enzyme function have been identified far from the luciferin binding site; 23 such critical residues are often revealed only by random mutagenesis approaches. 57–58 Screening libraries of completely random mutants was impractical in our case, though, owing to the large library sizes needed to achieve adequate enzyme coverage.…”

“…22 These same mutations also resulted in sharply reduced emission with D-luciferin, providing key precedent for the development and utilization of orthogonal pairs. 23 The mutant enzymes from these studies, though, were less selective for one analog over another perhaps due to the structural similarities between the luciferin scaffolds. Simultaneous enzyme-substrate manipulation has also been applied to aequorin (a marine photoprotein) and the luciferase from the deep-sea shrimp Oplophorus gracilirostris .…”

Brominated Luciferins Are Versatile Bioluminescent Probes

“…Fluc can tolerate a variety of modified substrates, although most are not processed as efficiently as d ‐luciferin, resulting in reduced photon production . Improved light outputs have been achieved, in some cases, by using mutant luciferases …”

Firefly-mimicking intensive and long-lasting chemiluminescence hydrogels

“…In the past, enzyme-involved CL reactions are main CL systems producing long-lasting emission. For enzymatic reactions such as firefly bioluminescence (BL) system, bacterial BL system, alkaline phosphatase-3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane system and luminol-H 2 O 2 -peroxidases system, long-lasting emission arose from the turnover of the enzymes and excessive substrates 7 – 9 . However, enzyme inactivation was the main reason for light decay in the above CL systems 10 .…”