Multicomponent Bioluminescence Imaging with a π-Extended Luciferin.

Bioluminescence imaging with luciferase-luciferin pairs is commonly used for monitoring biological processes in cells and whole organisms. Traditional bioluminescent probes are limited in scope, though, as they cannot be easily distinguished in biological environments, precluding efforts to visualize multicellular processes. Additionally, many luciferase-luciferin pairs emit light that is poorly tissue penetrant, hindering efforts to visualize targets in deep tissues. To address these issues, we synthesized a set of π-extended luciferins that were predicted to be red-shifted luminophores. The scaffolds were designed to be rotationally labile such that they produced light only when paired with luciferases capable of enforcing planarity. A luciferin comprising an intramolecular "lock" was identified as a viable light-emitting probe. Native luciferases were unable to efficiently process the analog, but a complementary luciferase was identified via Rosetta-guided enzyme design. The unique enzyme-substrate pair is red-shifted compared to well-known bioluminescent tools. The probe set is also orthogonal to other luciferase-luciferin probes and can be used for multicomponent imaging. Four substrate-resolved luciferases were imaged in a single session. Collectively, this work provides the first example of Rosetta-guided design in engineering bioluminescent tools and expands the scope of orthogonal imaging probes.

Bioluminescence imaging of carbon monoxide in living cells based on a selective deiodination reaction.

d-Luciferin is a popular bioluminescent substrate of luciferase in the presence of ATP. It is used in luciferase-based bioluminescence imaging and cell-based high-throughput screening applications. Herein, the iodination of d-luciferin was undertaken and explored as a bioluminescence probe without the need for light excitation to sensitively trace and image carbon monoxide (CO) in liver cancer cells. The bioluminescent probe (7'-iodo-luciferin) exhibited excellent selectivity for CO detection in vitro. This new probe could image exogenous and endogenous CO in the luciferase-transfected cancer cells. This new probe might be used for evaluating the roles of CO in various biological processes.

In vivo bioluminescence imaging of labile iron pools in a murine model of sepsis with a highly selective probe.

Iron plays an essential role in biological system. An approach for in vivo imaging of this metal ion is needed to investigate its complex contributions to physiological and pathological processes. Herein, we present a bioluminescent probe FP-1 as a powerful tool for targeting Fe2+ detection in vitro and in vivo. The turn-on sensing scheme is based on the caged strategy of luciferin-luciferase system. FP-1 not only can detect accumulations of exogenous Fe2+ in living animal, but also has the capability of monitoring labile endogenous Fe2+ levels in animal model of sepsis. Implementation of this technique provides a valuable opportunity for understanding underlying mechanisms of Fe2+ in biological processes and disease states.

Gram-scale synthesis of luciferins derived from coelenterazine and original insights into their bioluminescence properties.

An original gram-scale synthesis of O-acetylated forms of coelenterazine, furimazine or hydroxy-bearing analogues of luciferins is described. The comparison over two hours of their bioluminescence, using the nanoKAZ/NanoLuc luciferase, provides remarkable insights useful for the selection of a substrate adapted for a given application.

Bioorthogonal chemistry in bioluminescence imaging.

Bioorthogonal chemistry has developed significant over the past few decades, to the particular benefit of molecular imaging. Bioluminescence imaging (BLI) along with other imaging modalities have significantly benefitted from this chemistry. Here, we review bioorthogonal reactions that have been used to signific antly broaden the application range of BLI.

Visualization of mercury(ii) accumulation in vivo using bioluminescence imaging with a highly selective probe.

Mercury is a highly toxic environmental pollutant that negatively affects human health. Thus, an in vivo method for noninvasive imaging of mercury(ii) and visualization of its accumulation within living systems would be advantageous. Herein, we describe a reaction-based bioluminescent probe for detection of mercury(ii) in vitro and accumulation in vivo. The application of this probe would help to shed light on the intricate contributions of mercury(ii) to various physiological and pathological processes.

Lessons Learned from Luminous Luciferins and Latent Luciferases.

Compared to the broad palette of fluorescent molecules, there are relatively few structures that are competent to support bioluminescence. Here, we focus on recent advances in the development of luminogenic substrates for firefly luciferase. The scope of this light-emitting chemistry has been found to extend well beyond the natural substrate and to include enzymes incapable of luciferase activity with d-luciferin. The broadening range of luciferin analogues and evolving insight into the bioluminescent reaction offer new opportunities for the construction of powerful optical reporters of use in live cells and animals.

Bioluminescent probe for detecting endogenous hypochlorite in living mice.

As a kind of biologically important reactive oxygen species (ROS), hypochlorite (ClO-) plays a crucial role in many physiological processes. As such, endogenous ClO- is a powerful antibacterial agent during pathogen invasion. Nonetheless, excessive endogenous ClO- could pose a health threat to mammalian animals including humans. However, the detection of endogenous ClO- by bioluminescence probes in vivo remains a considerable challenge. Herein, based on a caged strategy, we developed a turn-on bioluminescent probe 1 for the highly selective detection of ClO-in vitro and imaging endogenous ClO- in a mouse inflammation model. We anticipate that such a probe could help us understand the role of endogenous ClO- in a variety of physiological and pathological processes.

A Dual-Color Bioluminescence Reporter Mouse for Simultaneous in vivo Imaging of T Cell Localization and Function.

Non-invasive imaging technologies to visualize the location and functionality of T cells are of great value in immunology. Here, we describe the design and generation of a transgenic mouse in which all T cells constitutively express green-emitting click-beetle luciferase (CBG99) while expression of the red-emitting firefly luciferase (PpyRE9) is induced by Nuclear Factor of Activated T cells (NFAT) such as during T cell activation, which allows multicolor bioluminescence imaging of T cell location and function. This dual-luciferase mouse, which we named TbiLuc, showed high constitutive luciferase expression in lymphoid organs such as lymph nodes and the spleen. Ex vivo purified CD8+ and CD4+ T cells both constitutively expressed luciferase, whereas B cells showed no detectable signal. We cross-bred TbiLuc mice to T cell receptor-transgenic OT-I mice to obtain luciferase-expressing naïve CD8+ T cells with defined antigen-specificity. TbiLuc*OT-I T cells showed a fully antigen-specific induction of the T cell activation-dependent luciferase. In vaccinated mice, we visualized T cell localization and activation in vaccine-draining lymph nodes with high sensitivity using two distinct luciferase substrates, D-luciferin and CycLuc1, of which the latter specifically reacts with the PpyRE9 enzyme. This dual-luciferase T cell reporter mouse can be applied in many experimental models studying the location and functional state of T cells.

In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection.

Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe2+ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe2+ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.