Coelenterazine Indicators for the Specific Imaging of Human and Bovine Serum Albumins.

Albumin assays in serum are important for the prognostic assessment of many life-threatening diseases, such as heart failure, liver disease, malnutrition, inflammatory bowel disease, infections, and kidney disease. In this study, synthetic coelenterazine (CTZ) indicators are developed to quantitatively illuminate human and bovine serum albumins (HSA and BSA) with high specificity. Their functional groups were chemically modified to specifically emit luminescence with HSA and BSA. The CTZ indicators were characterized by assaying the most abundant serum proteins and found that the CTZ indicators S6 and S6h were highly specific to HSA and BSA, respectively. Their colors were dramatically converted from blue, peaked at 480 nm, to yellowish green, peaked at 535 nm, according to the HSA-BSA mixing ratios, wherein the origins and mixing levels of the albumins can be easily determined by their colors and peak positions. The kinetic properties of HSA and BSA were investigated in detail, confirming that the serum albumins catalyze the CTZ indicators, which act as pseudo-luciferases. The catalytic reactions were efficiently inhibited by specific inhibitors, blocking the drug-binding sites I and II of HSA and BSA. Finally, the utility of the CTZ indicators was demonstrated through a quantitative imaging of the real fetal bovine serum (FBS). This study is the first example to show that the CTZ indicators specify HSA and BSA with different colors. This study contributes to the expansion of the toolbox of optical indicators, which efficiently assays serum proteins in physiological samples. Considering that these CTZ indicators immediately report quantitative optical signals with high specificity, they provide solutions to conventional technical hurdles on point-of-care assays of serum albumins.

Bright Molecular Strain Probe Templates for Reporting Protein-Protein Interactions.

Imaging protein-protein interactions (PPIs) is a hot topic in molecular medicine in the postgenomic sequencing era. In the present study, we report bright and highly sensitive single-chain molecular strain probe templates which embed full-length Renilla luciferase 8.6-535SG (RLuc86SG) or Artificial luciferase 49 (ALuc49) as reporters. These reporters were deployed between FKBP-rapamycin binding domain (FRB) and FK506-binding protein (FKBP) as a PPI model. This unique molecular design was conceptualized to exploit molecular strains of the sandwiched reporters appended by rapamycin-triggered intramolecular PPIs. The ligand-sensing properties of the templates were maximized by interface truncations and substrate modulation. The highest fold intensities, 9.4 and 16.6, of the templates were accomplished with RLuc86SG and ALuc49, respectively. The spectra of the templates, according to substrates, revealed that the colors are tunable to blue, green, and yellow. The putative substrate-binding chemistry and the working mechanisms of the probes were computationally modeled in the presence or absence of rapamycin. Considering that the molecular strain probe templates are applicable to other PPI models, the present approach would broaden the scope of the bioassay toolbox, which harnesses the privilege of luciferase reporters and the unique concept of the molecular strain probes into bioassays and molecular imaging.

S-Series Coelenterazine-Driven Combinatorial Bioluminescence Imaging Systems for Mammalian Cells.

A unique combinatorial bioluminescence (BL) imaging system was developed for determining molecular events in mammalian cells with various colors and BL intensity patterns. This imaging system consists of one or multiple reporter luciferases and a series of novel coelenterazine (CTZ) analogues named "S-series". For this study, ten kinds of novel S-series CTZ analogues were synthesized and characterized concerning the BL intensities, spectra, colors, and specificity of various marine luciferases. The characterization revealed that the S-series CTZ analogues luminesce with blue-to-orange-colored BL spectra with marine luciferases, where the most red-shifted BL spectrum peaked at 583 nm. The colors completed a visible light color palette with those of our precedent C-series CTZ analogues. The synthesized substrates S1, S5, S6, and S7 were found to have a unique specificity with marine luciferases, such as R86SG, NanoLuc (shortly, NLuc), and ALuc16. They collectively showed unique BL intensity patterns to identify the marine luciferases together with colors. The marine luciferases, R86SG, NLuc, and ALuc16, were multiplexed into multi-reporter systems, the signals of which were quantitatively unmixed with the specific substrates. When the utility was applied to a single-chain molecular strain probe, the imaging system simultaneously reported three different optical indexes for a ligand, i.e., unique BL intensity and color patterns for identifying the reporters, together with the ligand-specific fold intensities in mammalian cells. This study directs a new combinatorial BL imaging system to specific image molecular events in mammalian cells with multiple optical indexes.

C-Series Coelenterazine-Driven Bioluminescence Signature Imaging.

The present study introduces a unique BL signature imaging system with novel CTZ analogues named "C-series." Nine kinds of C-series CTZ analogues were first synthesized, and BL intensity patterns and spectra were then examined according to the marine luciferases. The results show that the four CTZ analogues named C3, C4, C6, and C7, individually or collectively luminesce with completely distinctive BL spectral signatures and intensity patterns according to the luciferases: Renilla luciferase (RLuc), NanoLuc, and artificial luciferase (ALuc). The signatural reporters were multiplexed into a multi-reporter system comprising RLuc8.6-535SG and ALuc16. The usefulness of the signatural reporters was further determined with a multi-probe system that consists of two single-chain probes embedding RLuc8 and ALuc23. This study is a great addition to the study of conventional bioassays with a unique methodology, and for the specification of each signal in a single- or multi-reporter system using unique BL signatures and patterns of reporter luciferases.

Multiplex quadruple bioluminescent assay system.

Bioluminescence (BL) is unique cold body radiation of light, generated by luciferin-luciferase reactions and commonly used in various bioassays and molecular imaging. However, most of the peak emissions of BL populate the blue-yellow region and have broad spectral bandwidths and thus superimpose each other, causing optical cross-leakages in multiplex assays. This study synthesized a new series of coelenterazine (CTZ) analogues, named K-series, that selectively illuminates marine luciferases with unique, blue-shifted spectral properties. The optical property and specificity of the K-series CTZ analogues were characterized by marine luciferases, with K2 and K5 found to specifically luminesce with ALuc- and RLuc-series marine luciferases, respectively. The results confirmed that the luciferase specificity and color variation of the CTZ analogues minimize the cross-leakages of BL signals and enable high-throughput screening of specific ligands in the mixture. The specificity and color variation of the substrates were further tailored to marine luciferases (or single-chain bioluminescent probes) to create a multiplex quadruple assay system with four integrated, single-chain bioluminescent probes, with each probe designed to selectively luminesce only with its specific ligand (first authentication) and a specific CTZ analogue (second authentication). This unique multiplex quadruple bioluminescent assay system is an efficient optical platform for specific and high-throughput imaging of multiple optical markers in bioassays without optical cross-leakages.

How to Select Firefly Luciferin Analogues for In Vivo Imaging.

Bioluminescence reactions are widely applied in optical in vivo imaging in the life science and medical fields. Such reactions produce light upon the oxidation of a luciferin (substrate) catalyzed by a luciferase (enzyme), and this bioluminescence enables the quantification of tumor cells and gene expression in animal models. Many researchers have developed single-color or multicolor bioluminescence systems based on artificial luciferin analogues and/or luciferase mutants, for application in vivo bioluminescence imaging (BLI). In the current review, we focus on the characteristics of firefly BLI technology and discuss the development of luciferin analogues for high-resolution in vivo BLI. In addition, we discuss the novel luciferin analogues TokeOni and seMpai, which show potential as high-sensitivity in vivo BLI reagents.