Abstract
Firefly luciferase-based ATP detection assays are frequently used as a sensitive, cost-efficient method for monitoring hygiene in many industrial settings. Solutions of detection reagent, containing a mixture of a substrate and luciferase enzyme that produces photons in the presence of ATP, are relatively unstable and maintain only a limited shelf life even under refrigerated conditions. It is therefore common for the individual performing a hygiene test to manually prepare fresh reagent at the time of monitoring. To simplify sample processing, a liquid detection reagent with improved thermal stability is needed. The engineered firefly luciferase, Ultra-Glo™, fulfills one aspect of this need and has been valuable for hygiene monitoring because of its high resistance to chemical and thermal inactivation. However, solutions containing both Ultra-Glo™ luciferase and its substrate luciferin gradually lose the ability to effectively detect ATP over time. We demonstrate here that dehydroluciferin, a prevalent oxidative breakdown product of luciferin, is a potent inhibitor of Ultra-Glo™ luciferase and that its formation in the detection reagent is responsible for the decreased ability to detect ATP. We subsequently found that dialkylation at the 5-position of luciferin (e.g., 5,5-dimethylluciferin) prevents degradation to dehydroluciferin and improves substrate thermostability in solution. However, since 5,5-dialkylluciferins are poorly utilized by Ultra-Glo™ luciferase as substrates, we used structural optimization of the luciferin dialkyl modification and protein engineering of Ultra-Glo™ to develop a luciferase/luciferin pair that shows improved total reagent stability in solution at ambient temperature. The results of our studies outline a novel luciferase/luciferin system that could serve as foundations for the next generation of bioluminescence ATP detection assays with desirable reagent stability.
Highlights
Bioluminescence is well established as a highly sensitive technology for probing biological systems [1,2,3,4,5]
It was hypothesized that replacement of H atoms at 5 positions in the luciferin structure with alkyl substituents could potentially block the oxidation of the thiazoline ring, preventing inhibitory dehydroluciferin accumulation, thereby improving overall assay stability and performance (Fig 1A)
We observed no degradation for III-a over the course of the study, whereas >50% of LH2 decomposed into dehydroluciferin under identical conditions (Fig 1B)
Summary
Bioluminescence is well established as a highly sensitive technology for probing biological systems [1,2,3,4,5]. Firefly luciferases and their substrate, D-luciferin (LH2), have been. Thermostable substrates for bioluminescence detection systems employees of Promega Corporation. Promega Corporation manufactures and sells Ultra-GloTM luciferase, luciferin, and ATP detection reagents. Ultra-GloTM luciferase mutations are disclosed in the published patent application US20200071682A1 “Luciferase Enzymes For Use With Thermostable Luciferins In Bioluminescent Assays”, and the luciferin analogs are disclosed in granted patent US 10,400,264 and the published patent application US 20190338340A1, “5,5disubstituted Luciferins And Their Use In Luciferase-based Assays” owned by Promega Corporation. The authors confirm that these competing interests do not alter their adherence to all the PLOS ONE policies on sharing data and materials
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