Brominated Luciferins Are Versatile Bioluminescent Probes

Steinhardt, Rachel C.; Rathbun, Colin M.; Krull, Brandon T.; Yu, Jason M.; Yang, Yuhang; Nguyen, Brian D.; Kwon, Jake; McCutcheon, David C.; Furche, Filipp; Prescher, Jennifer A.

doi:10.1002/cbic.201600564

Cited by 35 publications

(41 citation statements)

References 36 publications

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“…While only approximately 3% of the mutants were functional on 4’Br-Luc, compared to approximately 12% for D-luc, that is to be expected considering that Fluc displays 3% of the luminescence when using 4’Br-Luc than with D-luc. 30…”

Section: Resultsmentioning

confidence: 99%

Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases

et al. 2017

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Directed evolution has proven to be an invaluable tool for protein engineering; however, there is still a need for developing new approaches to continue to improve the efficiency and efficacy of these methods. Here, we demonstrate a new method for library design that applies a previously developed bioinformatic method, Statistical Coupling Analysis (SCA). SCA uses homologous enzymes to identify amino acid positions that are mutable and functionally important and engage in synergistic interactions between amino acids. We use SCA to guide a library of the protein luciferase and demonstrate that, in a single round of selection, we can identify luciferase mutants with several valuable properties. Specifically, we identify luciferase mutants that possess both red-shifted emission spectra and improved stability relative to those of the wild-type enzyme. We also identify luciferase mutants that possess a >50-fold change in specificity for modified luciferins. To understand the mutational origin of these improved mutants, we demonstrate the role of mutations at N229, S239, and G246 in altered function. These studies show that SCA can be used to guide library design and rapidly identify synergistic amino acid mutations from a small library.

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Section: Resultsmentioning

confidence: 99%

Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases

et al. 2017

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“…Next, we sought to evaluate the ability of our probes to serve as chemiluminescence agents for microscopy cell imaging.Chemiluminescence cell images have been obtained using luciferase-based probes and ac hemiluminescence bgalactosidase probe; [22,[32][33][34] however,i nb oth examples cells were transfected with the gene encoding the enzyme. Chemiluminescence microscopy cell-images were never demonstrated for natively-expressed endogenous enzymes,s uch as cathepsin B.…”

Section: Angewandte Chemiementioning

confidence: 99%

Unprecedented Sensitivity in a Probe for Monitoring Cathepsin B: Chemiluminescence Microscopy Cell‐Imaging of a Natively Expressed Enzyme

2017

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Until recently, chemiluminescence cell images could only be obtained using luciferase-activated probes. Moreover, chemiluminescence microscopy cell-imaging has not been demonstrated for natively expressed enzymes like cathepsin B. Herein, we describe the design, synthesis, and evaluation of the first chemiluminescence probe for the detection and imaging of cathepsin B. The probe activation mechanism relies on the release of a dioxetane intermediate, which undergoes chemiexcitation to emit green light with high efficiency under physiological conditions. Using the probe, we obtained clear images of cancerous leukemia and colon cells. This is the first demonstration of chemiluminescence cell images obtained by a probe for a natively expressed endogenous enzyme. We anticipate that the concept presented in this study will be broadly used to develop analogous probes for other important proteases relevant to biomolecular processes.

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“…This small molecule is the effective light emitter; thus, modifications to the scaffold can, in principle, impact both pharmacokinetic and spectral properties. There have been many notable successes in engineering unique luciferins in recent years . However, many of the scaffolds exhibit reduced photon outputs with luciferase, limiting their use in some cellular and tissue environments.…”

Section: Introductionmentioning

confidence: 99%

Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging

2018

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New applications for bioluminescence imaging require an expanded set of luciferase enzymes and luciferin substrates. Here, we report two novel luciferins for use in vitro and in cells. These molecules comprise regioisomeric pyridone cores that can be accessed from a common synthetic route. The analogues exhibited unique emission spectra with firefly luciferase, although photon intensities remained weak. Enhanced light outputs were achieved by using mutant luciferase enzymes. One of the luciferin-luciferase pairs produced light on par with native probes in live cells. The pyridone analogues and complementary luciferases add to a growing set of designer probes for bioluminescence imaging.

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Brominated Luciferins Are Versatile Bioluminescent Probes

Cited by 35 publications

References 36 publications

Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases

Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases

Unprecedented Sensitivity in a Probe for Monitoring Cathepsin B: Chemiluminescence Microscopy Cell‐Imaging of a Natively Expressed Enzyme

Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging

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