Multiplexed bioluminescence imaging with a substrate unmixing platform.

Bioluminescent tools can illuminate cellular features in whole organisms. Multi-component tracking remains challenging, though, owing to a lack of well-resolved probes and long imaging times. To address the need for more rapid, quantitative, and multiplexed bioluminescent readouts, we developed an analysis pipeline featuring sequential substrate administration and serial image acquisition. Light output from each luciferin is layered on top of the previous image, with minimal delay between substrate delivery. A MATLAB algorithm was written to analyze bioluminescent images generated from the rapid imaging protocol and deconvolute (i.e., unmix) signals from luciferase-luciferin pairs. Mixtures comprising three to five luciferase reporters were readily distinguished in under 50 min; this same experiment would require days using conventional workflows. We further showed that the algorithm can be used to accurately quantify luciferase levels in heterogeneous mixtures. Based on its speed and versatility, the multiplexed imaging platform will expand the scope of bioluminescence technology.

Rapid Multicomponent Bioluminescence Imaging via Substrate Unmixing.

Studies of biological function demand probes that can report on processes in real time and in physiological environments. Bioluminescent tools are uniquely suited for this purpose, as they enable sensitive imaging in cells and tissues. Bioluminescent reporters can also be monitored continuously over time without detriment, as excitation light is not required. Rather, light emission derives from luciferase-luciferin reactions. Several engineered luciferases and luciferins have expanded the scope of bioluminescence imaging in recent years. Multicomponent tracking remains challenging, though, due to a lack of streamlined methods to visualize combinations of bioluminescent reporters. Conventional approaches image one luciferase at a time. Consequently, short-term changes in cell growth or gene expression cannot be easily captured. Here, we report a strategy for rapid, multiplexed imaging with a wide range of luciferases and luciferins. Sequential addition of orthogonal luciferins, followed by substrate unmixing, enabled facile detection of multiple luciferases in vitro and in vivo. Multicomponent imaging in mice was also achieved on the minutes-to-hours time scale.

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

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.

Bioluminescent Probes for Imaging Biology beyond the Culture Dish.

Bioluminescence with luciferase-luciferin pairs is an attractive method for surveying cells in live tissues and whole organisms. Recent advances in luciferin chemistry and luciferase engineering are further expanding the scope of the technology. It is now possible to spy on cells in a variety of deep tissues and visualize multicellular interactions, feats that are enabling new questions to be asked and new ideas to be explored. This perspective piece highlights recent successes in bioluminescent probe development and their applications to imaging in live cells, tissues, and animals.

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.

Parallel Screening for Rapid Identification of Orthogonal Bioluminescent Tools.

Bioluminescence imaging with luciferase enzymes and luciferin small molecules is a well-established technique for tracking cells and other biological features in rodent models. Despite its popularity, bioluminescence has long been hindered by a lack of distinguishable probes. Here we present a method to rapidly identify new substrate-selective luciferases for multicomponent imaging. Our strategy relies on parallel screening of luciferin analogues with panels of mutant enzymes. The compiled data set is then analyzed in silico to uncover mutually orthogonal sets. Using this approach, we screened 159 mutant enzymes with 12 luciferins. Thousands of orthogonal pairs were revealed with sufficient selectivity for use in biological environments. Over 100 pairs were validated in vitro, and three were applied in cell and animal models. The parallel screening method is both generalizable and scalable and will streamline the search for larger collections of orthogonal probes.

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.

Design and Synthesis of an Alkynyl Luciferin Analogue for Bioluminescence Imaging.

Herein, the synthesis and characterization of an alkyne-modified luciferin is reported. This bioluminescent probe was accessed using C-H activation methodology and was found to be stable in solution and capable of light production with firefly luciferase. The luciferin analogue was also cell permeant and emitted more redshifted light than d-luciferin, the native luciferase substrate. Based on these features, the alkynyl luciferin will be useful for a variety of imaging applications.

Recognition and site-selective transformation of monosaccharides by using copper(II) catalysis.

We demonstrate copper(II)-catalyzed acylation and tosylation of monosaccharides. Various carbohydrate derivatives, including glucopyranosides and ribofuranosides, are obtained in high yields and regioselectivities. Using this versatile strategy, the site of acylation can be switched by choice of ligand. Preliminary mechanistic studies support nucleophilic addition of a copper-sugar complex to the acyl chloride to be turnover limiting.

Rate-limiting step of the Rh-catalyzed carboacylation of alkenes: C-C bond activation or migratory insertion?

Rhodium-catalyzed intramolecular carboacylation of alkenes, achieved using quinolinyl ketones containing tethered alkenes, proceeds via the activation and functionalization of a carbon-carbon single bond. This transformation has been demonstrated using RhCl(PPh(3))(3) and [Rh(C(2)H(4))(2)Cl](2) catalysts. Mechanistic investigations of these systems, including determination of the rate law and kinetic isotope effects, were utilized to identify a change in mechanism with substrate. With each catalyst, the transformation occurs via rate-limiting carbon-carbon bond activation for species with minimal alkene substitution, but alkene insertion becomes rate-limiting for more sterically encumbered substrates. Hammett studies and analysis of a series of substituted analogues provide additional insight into the nature of these turnover-limiting elementary steps of catalysis and the relative energies of the carbon-carbon bond activation and alkene insertion steps.

Rhodium-catalyzed acylation with quinolinyl ketones: carbon-carbon single bond activation as the turnover-limiting step of catalysis.

The rhodium-catalyzed intramolecular carboacylation of quinolinyl ketones serves as an ideal subject for the mechanistic study of carbon-carbon bond activation. Combined kinetic and NMR studies of this reaction allowed the identification of the catalytic resting state and determination of the rate law, (12)C/(13)C kinetic isotope effects, and activation parameters. These results have identified the activation of a ketone-arene carbon-carbon single bond as the turnover-limiting step of catalysis and provided quantitative detail into this process.