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Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase.

ABSTRACT: Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K⁺), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K⁺-binding luciferin named potassiorin. Additionally, we engineered a luciferase enzyme called BRIPO (bioluminescent red indicator for potassium) to work synergistically with potassiorin, resulting in optimized K⁺-dependent bioluminescence responses. Through extensive validation in cell lines, primary neurons, and live mice, we demonstrated the efficacy of this new tool for detecting K⁺. Our research demonstrates an innovative concept of incorporating sensory moieties into luciferins to modulate luciferase activity. This approach has great potential for developing a wide range of bioluminescent indicators, advancing bioluminescence imaging (BLI), and enabling the study of various analytes in biological systems.

SUBMITTER: Zhao S

PROVIDER: S-EPMC10980066 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase.

Zhao Shengyu S Xiong Ying Y Sunnapu Ranganayakulu R Zhang Yiyu Y Tian Xiaodong X Ai Hui-Wang HW

bioRxiv : the preprint server for biology 20240314

Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K<sup>+</sup>), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K<sup>+</sup>-binding luciferin named potassiori ...[more]

PMID: 38559024

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Project description:PurposeFirefly luciferase catalyzes the oxidative decarboxylation of D: -luciferin to oxyluciferin in the presence of cofactors, producing bioluminescence. This reaction is used in optical bioluminescence-based molecular imaging approaches to detect the expression of the firefly luciferase reporter gene. Biokinetics and distribution of the substrate most likely have a significant impact on levels of light signal and therefore need to be investigated.MethodsBenzene ring (14)C(U)-labeled D-luciferin was utilized. Cell uptake and efflux assays, murine biodistribution, autoradiography and CCD-camera based optical bioluminescence imaging were carried out to examine the in vitro and in vivo characteristics of the tracer in cell culture and in living mice respectively.ResultsRadiolabeled and unlabeled D-luciferin revealed comparable levels of light emission when incubated with equivalent amounts of the firefly luciferase enzyme. Cell uptake assays in pCMV-luciferase-transfected cells showed slow trapping of the tracer and relatively low uptake values (up to 22.9-fold higher in firefly luciferase gene-transfected vs. nontransfected cells, p = 0.0002). Biodistribution studies in living mice after tail-vein injection of (14)C-D-luciferin demonstrated inhomogeneous tracer distribution with early predominant high radioactivity levels in kidneys (10.6% injected dose [ID]/g) and liver (11.9% ID/g), followed at later time points by the bladder (up to 81.3% ID/g) and small intestine (6.5% ID/g), reflecting the elimination routes of the tracer. Kinetics and uptake levels profoundly differed when using alternate injection routes (intravenous versus intraperitoneal). No clear trapping of (14)C-D-luciferin in firefly luciferase-expressing tissues could be observed in vivo.ConclusionsThe data obtained with (14)C-D-luciferin provide insights into the dynamics of D: -luciferin cell uptake, intracellular accumulation, and efflux. Results of the biodistribution and autoradiographic studies should be useful for optimizing and adapting optical imaging protocols to specific experimental settings when utilizing the firefly luciferase and D: -luciferin system.

Dataset Information

Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase.

Publications

Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase.

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