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

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.

Coelenterazine-Type Bioluminescence-Induced Optical Probes for Sensing and Controlling Biological Processes.

Bioluminescence-based probes have long been used to quantify and visualize biological processes in vitro and in vivo. Over the past years, we have witnessed the trend of bioluminescence-driven optogenetic systems. Typically, bioluminescence emitted from coelenterazine-type luciferin-luciferase reactions activate light-sensitive proteins, which induce downstream events. The development of coelenterazine-type bioluminescence-induced photosensory domain-based probes has been applied in the imaging, sensing, and control of cellular activities, signaling pathways, and synthetic genetic circuits in vitro and in vivo. This strategy can not only shed light on the mechanisms of diseases, but also promote interrelated therapy development. Here, this review provides an overview of these optical probes for sensing and controlling biological processes, highlights their applications and optimizations, and discusses the possible future directions.

A bioluminescent probe for longitudinal monitoring of mitochondrial membrane potential.

Mitochondrial membrane potential (ΔΨm) is a universal selective indicator of mitochondrial function and is known to play a central role in many human pathologies, such as diabetes mellitus, cancer and Alzheimer's and Parkinson's diseases. Here, we report the design, synthesis and several applications of mitochondria-activatable luciferin (MAL), a bioluminescent probe sensitive to ΔΨm, and partially to plasma membrane potential (ΔΨp), for non-invasive, longitudinal monitoring of ΔΨm in vitro and in vivo. We applied this new technology to evaluate the aging-related change of ΔΨm in mice and showed that nicotinamide riboside (NR) reverts aging-related mitochondrial depolarization, revealing another important aspect of the mechanism of action of this potent biomolecule. In addition, we demonstrated application of the MAL probe for studies of brown adipose tissue (BAT) activation and non-invasive in vivo assessment of ΔΨm in animal cancer models, opening exciting opportunities for understanding the underlying mechanisms and for discovery of effective treatments for many human pathologies.

Applications of bioluminescence in biotechnology and beyond.

Bioluminescence is the fascinating natural phenomenon by which living creatures produce light. Bioluminescence occurs when the oxidation of a small-molecule luciferin is catalysed by an enzyme luciferase to form an excited-state species that emits light. There are over 30 known bioluminescent systems but the luciferin-luciferase pairs of only 11 systems have been characterised to-date, whilst other novel systems are currently under investigation. The different luciferin-luciferase pairs have different light emission wavelengths and hence are suitable for various applications. The last decade or so has seen great advances in protein engineering, synthetic chemistry, and physics which have allowed luciferins and luciferases to reach previously uncharted applications. The bioluminescence reaction is now routinely used for gene assays, the detection of protein-protein interactions, high-throughput screening (HTS) in drug discovery, hygiene control, analysis of pollution in ecosystems and in vivo imaging in small mammals. Moving away from sensing and imaging, the more recent highlights of the applications of bioluminescence in biomedicine include the bioluminescence-induced photo-uncaging of small-molecules, bioluminescence based photodynamic therapy (PDT) and the use of bioluminescence to control neurons. There has also been an increase in blue-sky research such as the engineering of various light emitting plants. This has led to lots of exciting multidisciplinary science across various disciplines. This review focuses on the past, present, and future applications of bioluminescence. We aim to make this review accessible to all chemists to understand how these applications were developed and what they rely upon, in simple understandable terms for a graduate chemist.

A New Method for Albuminuria Measurement Using a Specific Reaction between Albumin and the Luciferin of the Firefly Squid Watasenia scintillans.

This study demonstrates that the luciferin of the firefly squid Watasenia scintillans, which generally reacts with Watasenia luciferase, reacted with human albumin to emit light in proportion to the albumin concentration. The luminescence showed a peak wavelength at 540 nm and was eliminated by heat or protease treatment. We used urine samples collected from patients with diabetes to quantify urinary albumin concentration, which is essential for the early diagnosis of diabetic nephropathy. Consequently, we were able to measure urinary albumin concentrations by precipitating urinary proteins with acetone before the reaction with luciferin. A correlation was found with the result of the immunoturbidimetric method; however, the Watasenia luciferin method tended to produce lower albumin concentrations. This may be because the Watasenia luciferin reacts with only intact albumin. Therefore, the quantification method using Watasenia luciferin is a new principle of urinary albumin measurement that differs from already established methods such as immunoturbidimetry and high-performance liquid chromatography.

Comparison of Bioluminescent Substrates in Natural Infection Models of Neglected Parasitic Diseases.

The application of in vivo bioluminescent imaging in infectious disease research has significantly increased over the past years. The detection of transgenic parasites expressing wildtype firefly luciferase is however hampered by a relatively low and heterogeneous tissue penetrating capacity of emitted light. Solutions are sought by using codon-optimized red-shifted luciferases that yield higher expression levels and produce relatively more red or near-infrared light, or by using modified bioluminescent substrates with enhanced cell permeability and improved luminogenic or pharmacokinetic properties. In this study, the in vitro and in vivo efficacy of two modified bioluminescent substrates, CycLuc1 and AkaLumine-HCl, were compared with that of D-luciferin as a gold standard. Comparisons were made in experimental and insect-transmitted animal models of leishmaniasis (caused by intracellular Leishmania species) and African trypanosomiasis (caused by extracellular Trypanosoma species), using parasite strains expressing the red-shifted firefly luciferase PpyRE9. Although the luminogenic properties of AkaLumine-HCl and D-luciferin for in vitro parasite detection were comparable at equal substrate concentrations, AkaLumine-HCl proved to be unsuitable for in vivo infection follow-up due to high background signals in the liver. CycLuc1 presented a higher in vitro luminescence compared to the other substrates and proved to be highly efficacious in vivo, even at a 20-fold lower dose than D-luciferin. This efficacy was consistent across infections with the herein included intracellular and extracellular parasitic organisms. It can be concluded that CycLuc1 is an excellent and broadly applicable alternative for D-luciferin, requiring significantly lower doses for in vivo bioluminescent imaging in rodent models of leishmaniasis and African trypanosomiasis.

Luciferase-free Luciferin Electrochemiluminescence.

Luciferin is one of Nature's most widespread luminophores, and enzymes that catalyze luciferin luminescence are the basis of successful commercial "glow" assays for gene expression and metabolic ATP formation. Herein we report an electrochemical method to promote firefly's luciferin luminescence in the absence of its natural biocatalyst-luciferase. We have gained experimental and computational insights on the mechanism of the enzyme-free luciferin electrochemiluminescence, demonstrated its spectral tuning from green to red by means of electrolyte engineering, proven that the colour change does not require, as still debated, a keto/enol isomerization of the light emitter, and gained evidence of the electrostatic-assisted stabilization of the charge-transfer excited state by double layer electric fields. Luciferin's electrochemiluminescence, as well as the in situ generation of fluorescent oxyluciferin, are applied towards an optical measurement of diffusion coefficients.

Orthogonal Bioluminescent Probes from Disubstituted Luciferins.

Bioluminescence imaging with luciferase-luciferin pairs is routinely used to monitor cellular functions. Multiple targets can be visualized in tandem using luciferases that process unique substrates, but only a handful of such orthogonal probes are known. Multiplexed studies require additional robust, light-emitting molecules. In this work, we report new luciferins for orthogonal imaging that comprise disubstituted cores. These probes were found to be bright emitters with various engineered luciferases. The unique patterns of light output also provided insight into enzyme-substrate interactions necessary for productive emission. Screening studies identified mutant luciferases that could preferentially process the disubstituted analogues, enabling orthogonal imaging with existing bioluminescent reporters. Further mutational analyses revealed the origins of substrate selectivity. Collectively, this work provides insights into luciferase-luciferin features relevant to bioluminescence and expands the number of probes for multicomponent tracking.

A novel yellow fluorescent protein of recombinant apoPholasin with dehydrocoelenterazine.

Pholasin is classified as a photoprotein and comprises apoPholasin (an apoprotein of pholasin) and an unknown prosthetic group as the light-emitting source. The luminescence reaction of pholasin is triggered by reactive oxygen species. Recombinant apoPholasin was recently expressed as a fusion protein of glutathione S-transferase (GST-apoPholasin) and purified from E. coli cells. By incubating non-fluorescent dehydrocoelenterazine (dCTZ, dehydrogenated form of CTZ) with GST-apoPholasin, the complex of GST-apoPholasin and dCTZ (GST-apoPholasin/dCTZ complex) was formed immediately and showed bright yellow fluorescence (λmax = 539 nm, excited at 430 nm). Unexpectedly, the fluorescent chromophore of the GST-apoPholasin/dCTZ complex was identified as non-fluorescent dCTZ. The luminescence intensity of the GST-apoPholasin/dCTZ complex was increased in a catalase-H2O2 system, but not in sodium hypochlorite.

Red-shifted luciferase-luciferin pairs for enhanced bioluminescence imaging.

Red-shifted bioluminescence reporters are desirable for biological imaging. We describe the development of red-shifted luciferins based on synthetic coelenterazine analogs and corresponding mutants of NanoLuc that enable bright bioluminescence. One pair in particular showed superior in vitro and in vivo sensitivity over commonly used bioluminescence reporters. We adapted this pair to develop a bioluminescence resonance-energy-based Antares reporter called Antares2, which offers improved signal from deep tissues.

Building Biological Flashlights: Orthogonal Luciferases and Luciferins for in Vivo Imaging.

Bioluminescence is widely used for real-time imaging in living organisms. This technology features a light-emitting reaction between enzymes (luciferases) and small molecule substrates (luciferins). Photons produced from luciferase-luciferin reactions can penetrate through heterogeneous tissue, enabling readouts of physiological processes. Dozens of bioluminescent probes are now available and many are routinely used to monitor cell proliferation, migration, and gene expression patterns in vivo. Despite the ubiquity of bioluminescence, traditional applications have been largely limited to imaging one biological feature at a time. Only a handful of luciferase-luciferin pairs can be easily used in tandem, and most are poorly resolved in living animals. Efforts to develop spectrally distinct reporters have been successful, but multispectral imaging in large organisms remains a formidable challenge due to interference from surrounding tissue. Consequently, a lack of well-resolved probes has precluded multicomponent tracking. An expanded collection of bioluminescent probes would provide insight into processes where multiple cell types drive physiological tasks, including immune function and organ development. We aimed to expand the bioluminescent toolkit by developing substrate-resolved imaging agents. The goal was to generate multiple orthogonal (i.e., noncross-reactive) luciferases that are responsive to unique scaffolds and could be used concurrently in living animals. We adopted a parallel engineering approach to genetically modify luciferases to accept chemically modified luciferins. When the mutants and analogs are combined, light is produced only when complementary enzyme-substrate partners interact. Thus, the pairs can be distinguished based on substrate selectivity, regardless of the color of light emitted. Sequential administration of the luciferins enables the unique luciferases to be illuminated (and thus resolved) within complex environments, including whole organisms. This Account describes our efforts to develop orthogonal bioluminescent probes, crafting custom luciferases (or "biological flashlights") that can selectively process luciferin analogs (or "batteries") to produce light. In the first section, we describe synthetic methods that were key to accessing diverse luciferin architectures. The second section focuses on identifying complementary luciferase enzymes via a combination of mutagenesis and screening. To expedite the search for orthogonal enzymes and substrates, we developed a computational algorithm to sift through large data sets. The third section features examples of the parallel engineering approach. We identified orthogonal enzyme-substrate pairs comprising two different classes of luciferins. The probes were vetted both in cells and whole organisms. This expanded collection of imaging agents is applicable to studies of immune function and other multicomponent processes. The final section of the Account highlights ongoing work toward building better bioluminescent tools. As ever-brighter and more selective probes are developed, the frontiers of what we can "see" in vivo will continue to expand.

Expression of recombinant apopholasin using a baculovirus-silkworm multigene expression system and activation via dehydrocoelenterazine.

Pholasin is a photoprotein derived from the glowing bivalve mollusk, Pholas dactylus. Even though the chemical structure of the prosthetic group (chromophore) responsible for the light emission character of the mollusk remains unknown, research has shown that the presence of dehydrocoelenterazine (DCL) increased light emission and that the dithiothreitol adduct of DCL was isolated from Pholasin®. To date, our research has been focused on activating apopholasin, the naturally occurring apoprotein of Pholasin®, using DCL. In the current study, the expression of recombinant apopholasin via a baculovirus-silkworm multigene expression system is reported. Additionally, the purification of apopholasin using a Flag®-affinity column, the activation of apopholasin using DCL, and the initiation of its luminescent character through the addition of a peroxidase-hydrogen peroxide mixture are reported. The peroxidase-H2O2-dependent luminescence was observed from the recombinant apopholasin activated with DCL.

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications.

: The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate-coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization-Ca2+-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme-substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases.

Mechanically Assisted Bioluminescence with Natural Luciferase.

Mechanochemical analogues have recently been established for several enzymatic reactions, but they require periodic interruption of the reaction for sampling, dissolution, and (bio)chemical analysis to monitor their progress. By applying a mechanochemical procedure to induce bioluminescence analogous to that used by the marine ostracod Cypridina (Vargula) hilgendorfii, here we demonstrate that the light emitted by a bioluminescent reaction can be used to directly monitor the progress of a mechanoenzymatic reaction without sampling. Mechanical treatment of Cypridina luciferase with luciferin generates bright blue light which can be readily detected and analyzed spectroscopically. This mechanically assisted bioluminescence proceeds through a mechanism identical to that of bioluminescence in solution, but has higher activation energy due to being diffusion-controlled in the viscous matrix. The results suggest that luciferases could be used as light-emissive reporters of mechanoenzymatic reactions.

Luciferin Regeneration in Firefly Bioluminescence via Proton-Transfer-Facilitated Hydrolysis, Condensation and Chiral Inversion.

Firefly bioluminescence is produced via luciferin enzymatic reactions in luciferase. Luciferin has to be unceasingly replenished to maintain bioluminescence. How is the luciferin reproduced after it has been exhausted? In the early 1970s, Okada proposed the hypothesis that the oxyluciferin produced by the previous bioluminescent reaction could be converted into new luciferin for the next bioluminescent reaction. To some extent, this hypothesis was evidenced by several detected intermediates. However, the detailed process and mechanism of luciferin regeneration remained largely unknown. For the first time, we investigated the entire process of luciferin regeneration in firefly bioluminescence by density functional theory calculations. This theoretical study suggests that luciferin regeneration consists of three sequential steps: the oxyluciferin produced from the last bioluminescent reaction generates 2-cyano-6-hydroxybenzothiazole (CHBT) in the luciferin regenerating enzyme (LRE) via a hydrolysis reaction; CHBT combines with L-cysteine in vivo to form L-luciferin via a condensation reaction; and L-luciferin inverts into D-luciferin in luciferase and thioesterase. The presently proposed mechanism not only supports the sporadic evidence from previous experiments but also clearly describes the complete process of luciferin regeneration. This work is of great significance for understanding the long-term flashing of fireflies without an in vitro energy supply.

2-S-cysteinylhydroquinone is an intermediate for the firefly luciferin biosynthesis that occurs in the pupal stage of the Japanese firefly, Luciola lateralis.

Firefly luciferin is a natural product that is well-known to function as the substrate of the bioluminescence reaction in luminous beetles. However, the details of the biosynthetic system are still unclear. In this study, we showed by LC-MS/MS analysis that stable isotope-labeled 2-S-cysteinylhydroquinone was incorporated into firefly luciferin in living firefly specimens. Comparison of the incorporation efficiency among the developmental stages suggested that firefly luciferin is biosynthesized predominantly in the pupal stage. We also accomplished the in vitro biosynthesis of firefly luciferin using 2-S-cysteinylhydroquinone and the crude buffer extract of firefly pupae, suggesting the presence of a biosynthetic enzyme in the pupal extract.

A Nanobionic Light-Emitting Plant.

The engineering of living plants for visible light emission and sustainable illumination is compelling because plants possess independent energy generation and storage mechanisms and autonomous self-repair. Herein, we demonstrate a plant nanobionic approach that enables exceptional luminosity and lifetime utilizing four chemically interacting nanoparticles, including firefly luciferase conjugated silica (SNP-Luc), d-luciferin releasing poly(lactic-co-glycolic acid) (PLGA-LH2), coenzyme A functionalized chitosan (CS-CoA) and semiconductor nanocrystal phosphors for longer wavelength modulation. An in vitro kinetic model incorporating the release rates of the nanoparticles is developed to maximize the chemiluminescent lifetimes to exceed 21.5 h. In watercress (Nasturtium officinale) and other species, the nanoparticles circumvent limitations such as luciferin toxicity above 400 µM and colocalization of enzymatic reactions near high adenosine triphosphate (ATP) production. Pressurized bath infusion of nanoparticles (PBIN) is introduced to deliver a mixture of nanoparticles to the entire living plant, well described using a nanofluidic mathematical model. We rationally design nanoparticle size and charge to control localization within distinct tissues compartments with 10 nm nanoparticles localizing within the leaf mesophyll and stomata guard cells, and those larger than 100 nm segregated in the leaf mesophyll. The results are mature watercress plants that emit greater than 1.44 × 1012 photons/sec or 50% of 1 µW commercial luminescent diodes and modulate "off" and "on" states by chemical addition of dehydroluciferin and coenzyme A, respectively. We show that CdSe nanocrystals can shift the chemiluminescent emission to 760 nm enabling near-infrared (nIR) signaling. These results advance the viability of nanobionic plants as self-powered photonics, direct and indirect light sources.

Orthogonal Luciferase-Luciferin Pairs for Bioluminescence Imaging.

Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate "hits" were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.

cybLuc: An Effective Aminoluciferin Derivative for Deep Bioluminescence Imaging.

To enhance the efficiency of firefly luciferase/luciferin bioluminescence imaging, a series of N-cycloalkylaminoluciferins (cyaLucs) were developed by introducing lipophilic N-cycloalkylated substitutions. The experimental results demonstrate that these cyaLucs are effective substrates for native firefly luciferase (Fluc) and can produce elevated bioluminescent signals in vitro, in cellulo, and in vivo. It should be noted that, in animal studies, N-cyclobutylaminoluciferin (cybLuc) at 10 µM (0.1 mL), which is 0.01% of the standard dose of d-luciferin (dLuc) used in mouse imaging, can radiate 20-fold more bioluminescent light than d-luciferin (dLuc) or aminoluciferin (aLuc) at the same concentration. Longer in vivo emission imaging using cybLuc suggests that it can be used for long-time observation. Regarding the mechanism of cybLuc, our cocrystal structure data from firefly luciferase with oxidized cybLuc suggested that oxidized cybLuc fits into the same pocket as oxyluciferin. Most interestingly, our results demonstrate that the sensitivity of cybLuc in brain tumor imaging contributes to its extended application in deep tissues.

Brominated Luciferins Are Versatile Bioluminescent Probes.

We report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed by using a common synthetic route. All analogues produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed by computation and photophysical measurements. The brightest brominated luciferin was further evaluated in cell and animal models. At low doses, the analogue outperformed the native substrate in cells. The remaining luciferins, although weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.